Cells producing glycoproteins having altered glycosylation patterns and method and use thereof

ABSTRACT

The disclosure relates to the field of glyco-engineering, more specifically, to eukaryotic cells wherein both an endoglucosaminidase and a glycoprotein are present. These cells can be used to deglycosylate or partly deglycosylate the (exogenous) glycoprotein, in particular, without the need for adding an extra enzyme. Methods are also provided for the application of these cells in protein production. According to one specific aspect, the eukaryotic cells are glyco-engineered yeast cells in which, additionally, at least one exogenous enzyme needed for complex glycosylation is present, e.g., allowing easier separation of differentially glycosylated glycoproteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 12/737,719, filed Apr. 8, 2011, now U.S. Pat. No. ______, whichapplication is a national phase entry under 35 U.S.C. §371 ofInternational Patent Application PCT/EP2009/060348, filed Aug. 10, 2009,designating the United States of America and published in English asInternational Patent Publication WO2010/015722 A1 on Feb. 11, 2010,which claims the benefit under Article 8 of the Patent CooperationTreaty to European Patent Application Serial Nos. 08162059.3 and08162063.5, both filed Aug. 8, 2008, the disclosure of each of which ishereby incorporated herein in its entirety by this reference.

TECHNICAL FIELD

The disclosure relates generally to the field of biotechnology andglyco-engineering, more specifically to eukaryotic cells, wherein bothan endoglucosaminidase and a glycoprotein are present. These cells canbe used to deglycosylate or partly deglycosylate the (exogenous)glycoprotein, in particular, without the need for adding an extraenzyme. Methods are also provided for the application of these cells inprotein production. Also envisaged herein is the particular subset ofglyco-engineered yeast cells, i.e., yeast cells having at least oneexogenous enzyme needed for complex glycosylation in addition to theendoglucosaminidase and the glycoprotein. These cells are particularlyuseful in providing more homogeneous or easily separable populations ofthe glycoprotein, which helps considerably in isolating onlyglycosylated proteins with the desired properties.

BACKGROUND

Glycoproteins are an important class of biomolecules that play crucialroles in many biological events such as cell adhesion, tumor metastasis,pathogen infection, and immune response. Most mammalian cell surfaceproteins and human serum proteins are glycoproteins and it is notsurprising then that therapeutic glycoproteins are an important class ofbiotechnology products. These include, amongst many others, granulocytemacrophage colony-stimulating factor, tissue plasminogen activator,interleukin-2, erythropoietin (EPO), and antibodies. Both natural andrecombinant glycoproteins are typically produced as a mixture ofglycoforms that differ only in the structure of the pendentoligosaccharides. This heterogeneity in glycosylation is a major problemin structural and functional studies of glycoproteins (e.g.,crystallization studies), as well as in development of glycoproteindrugs. The attached sugar chains may, for instance, have profoundeffects on protein folding, stability, action, pharmacokinetics, andserum half-life of the glycoprotein, and some sugar chains are veryimmunogenic.

Glycosylation is one of the most common post-translational modificationsof proteins in eukaryotes. N-glycosylation is a highly conservedmetabolic process, which in eukaryotes is essential for viability.Protein N-glycosylation originates in the endoplasmic reticulum (ER),where an N-linked oligosaccharide (Glc₃Man₉GlcNAc₂) assembled ondolichol (a lipid carrier intermediate) is transferred to theappropriate asparagines residue (Asn) of a nascent protein. This is aco-translational event largely common to all eukaryotic organisms. Thethree glucose residues and one specific α-1,2-linked mannose residue areremoved by specific glucosidases and an α-1,2-mannosidase in the ER,resulting in the core oligosaccharide structure, Man₈GlcNAc₂. Proteinswith this core sugar structure are transported to the Golgi apparatuswhere the sugar moiety undergoes various modifications.Glycosyltransferases and mannosidases line the inner (luminal) surfaceof the ER and Golgi apparatus and thereby provide a catalytic surfacethat allows for the sequential processing of glycoproteins as theyproceed through the ER and Golgi network. The multiple compartments ofthe cis, medial, and trans Golgi and the trans-Golgi Network (TGN),provide the different localities in which the ordered sequence ofglycosylation reactions can take place. As a glycoprotein proceeds fromsynthesis in the ER to full maturation in the late Golgi or TGN, it issequentially exposed to different glycosidases, mannosidases andglycosyltransferases, such that a specific N-glycan structure may besynthesized. There are significant differences in the modifications ofthe sugar chain in the Golgi apparatus between lower and highereukaryotes.

In higher eukaryotes, the N-linked oligosaccharides are typically highmannose, complex and mixed (hybrid) types of structures that varysignificantly from those produced in yeast (Kornfeld et al., Ann. Rev.Biochem. 54:631-664 (1985)). In mammalian cells, the modification of thesugar chain can follow three different pathways depending on the proteinmoiety to which it is added. That is: (1) the core sugar chain does notchange; (2) the core sugar chain is changed by adding theN-acetylglucosamine-1-phosphate moiety (GlcNAc-1-P) in UDP-N-acetylglucosamine (UDP-GlcNAc) to the 6-position of mannose in the core sugarchain, followed by removal of the GlcNAc moiety to form an acidic sugarchain in the glycoprotein; and (3) the core sugar chain is firstconverted into Man₅GlcNAc₂ by removing three mannose residues with Golgiα-Mannosidase I; Man₅GlcNAc₂ is then further modified by adding GlcNAcand removing two more mannose residues, followed by sequentially addingGlcNAc, galactose (Gal), GalNAc, fucose and N-acetylneuraminic acid(also called sialic acid (NeuNAc)) to form various hybrid or complexsugar chains (R. Kornfeld and S. Kornfeld, 1985; Chiba et al., 1998).Different organisms provide different glycosylation enzymes(glycosyltransferases and glycosidases) and different glycosylsubstrates, so that the final composition of a sugar side chain may varymarkedly depending upon the higher eukaryotic host. Typically, theprotein N glycans of animal glycoproteins have bi-, tri-, ortetra-antennary structures. These branched structures are synthesized bythe GlcNAc transferase-catalyzed addition of GlcNAc to regions of theoligosaccharide residue. Subsequent to their formation, the antennarystructures are terminated with different sugars including Gal, GalNAc,GlcNAc, fucose (Fuc) and sialic acid residues.

In yeast and filamentous fungi (lower eukaryotes), only a part of theMan₈₍₉₎GlcNAc₂ structures are (partially) trimmed down to Man₅GlcNAc₂.These oligosaccharides can then be further modified to fungal-specificglycans through the addition of mannose and/or mannosephosphate residuesin a diester linkage. The resulting glycans are known as “high-mannose”type glycans or mannans. For example, yeast glycopeptides includeoligosaccharide structures that consist of a high mannose core of 9-13mannose residues, or extended branched mannan outer chains consisting ofup to 200 residues (Ballou et al., Dev. Biol. 166:363-379 (1992);Trimble et al., Glycobiology 2:57-75 (1992)).

Considerable effort has been directed toward the identification andoptimization of new strategies for the preparation of glycopeptides andglycoproteins for therapeutic application. Probably the most documentedapproach amongst the many promising methods is the engineering ofcellular hosts that produce glycopeptides having a desired glycosylationpattern. For a recent review on how this can be achieved, in particularin yeast, see Wildt et al., Nature Reviews 2005, 119-28; and Hamilton etal., Curr. Opin. Biotechnol. 2007, 18(5):387-92. Other exemplary methodsinclude chemical synthesis, enzymatic synthesis, enzymatic remodeling offormed glycopeptides and, of course, methods that are hybrids orcombinations of one or more of these techniques.

Regarding cell host systems, in principle, mammalian, insect, yeast,fungal, plant or prokaryotic cell culture systems, can be used forproduction of most therapeutic and other glycopeptides in commerciallyfeasible quantities. In practice, however, a desired glycosylationpattern on a recombinantly produced protein is difficult to achieve. Forexample, bacteria do not N-glycosylate via the dolichol pathway, andyeast only produces oligomannose-type N-glycans, which are not generallyfound in large quantities in humans. Similarly, plant cells do notproduce sialylated oligosaccharides, a common constituent of humanglycopeptides. In addition, plants add xylose and/or α-1,3-linked fucoseto protein N-glycans, resulting in glycoproteins that differ instructure from animals and are immunogenic in mammals (Lerouge et al.,Plant Mol. Biol. 1998, 38(1-2):31-48; Betenbaugh et al., Curr. Opin.Struct. Biol. 2004, 14(5):601-6; Altmann, Int. Arch. Allergy Immunol.2007, 142(2):99-115). As recently reviewed, none of the insect cellsystems presently available for the production of recombinant mammalianglycopeptides will produce glycopeptides with the same glycans normallyfound when they are produced in mammals (Harrison and Jarvis, 2006,159). Moreover, glycosylation patterns of recombinant glycopeptides mayalso differ when produced under different cell culture conditions(Watson et al., Biotechnol. Prog. 10:39-44 (1994); and Gawlitzek et al.,Biotechnol. J. 42:117-131 (1995)) or even between glycopeptides producedunder nominally identical cell culture conditions in two differentbioreactors (Kunkel et al., Biotechnol. Prog. 2000:462-470 (2000)).

Thus, despite significant advances in this field, heterogeneity ofglycosylation remains an issue. Heterogeneity in the glycosylation ofrecombinantly produced glycopeptides arises because the cellularmachinery (e.g., glycosyltransferases and glycosidases) may vary fromspecies to species, cell to cell, or even from individual to individual.The substrates recognized by the various enzymes may be sufficientlydifferent that glycosylation may not occur at some sites or may bevastly modified from that of the native protein. Glycosylation ofrecombinant proteins produced in heterologous eukaryotic hosts willoften differ from the native protein. Therapeutic glycoproteins aretypically produced in cell culture systems as a mixture of glycoformsthat possess the same peptide backbone but differ in both the nature andsite of glycosylation. The heterogeneity in glycosylation posessignificant difficulty for the purification, efficacy, as well astherapeutic safety of glycoproteins. Cell and/or glyco-engineering andsome biochemical modifications may have yielded cells or (e.g., yeast)strains that produce recombinant glycoproteins with predominantglycoforms but, in most cases, as with natively expressed glycoproteins,the structures that have been obtained remain heterogeneous. Notably,different glycosylation forms can exert significantly different effectson the properties of a given protein, and some glycoforms can even causeallergy problems and undesired immune responses. This is, e.g.,particularly true for the high-mannose-type glycoproteins normallyproduced in yeast. Isolation of a glycoprotein having a particularglycosylation state from such a mixture of glycosylation forms isextremely difficult. However, as small amounts of impurities candramatically interfere with the desired activities of the glycoproteinof interest, such inhibition is also highly desirable.

In addition to preparing properly glycosylated glycopeptides byengineering the host cell to include the necessary compliment ofenzymes, efforts have been directed to the development of both de novosynthesis of glycopeptides and the in vitro enzymatic methods oftailoring the glycosylation of glycopeptides. Although great advanceshave been made in recent years in both carbohydrate chemistry and thesynthesis of glycopeptides (Arsequell et al., Tetrahedron:Assymetry10:3045 (1999)), there are still substantial difficulties associatedwith chemical synthesis of glycopeptides, particularly with theformation of the ubiquitous β-1,2-cis-mannoside linkage found inmammalian oligosaccharides. Moreover, regio- and stereo-chemicalobstacles must be resolved at each step of the de novo synthesis of acarbohydrate.

As enzyme-based syntheses have the advantages of regioselectivity andstereoselectivity, the use of enzymes to synthesize the carbohydrateportions of glycopeptides is a promising approach to preparingglycopeptides. Moreover, enzymatic syntheses can be performed usingunprotected substrates. Three principal classes of enzymes are used inthe synthesis of carbohydrates, glycosyltransferases (e.g.,N-acetylglucosaminyltransferases, oligosaccharyltransferases,sialyltransferases), glycoaminidases (e.g., PNGase F) and glycosidases.The glycosidases are further classified as exoglycosidases (e.g.,p-mannosidase, p-glucosidase), and endoglycosidases (e.g., Endo-A,Endo-M). Each of these classes of enzymes has been successfully usedsynthetically to prepare carbohydrates and glycoproteins. As an example,RNase B has been synthesized as a high-mannose glycosylated protein,after which the oligosaccharide was enzymatically removed (apart from asingle GlcNAc) and the correct glycoform was produced in subsequenttransglycosylation reactions using different enzymes (Witte et al., J.Am. Chem. Soc., 119:(9)2114-2118, 1997). More examples of howtransglycosylation may be used in glycoprotein synthesis are reviewedand described in Crout et al., Curr. Opin. Chem. Biol. 2:98-111 (1998);Arsequell, Tetrahedron:Assymetry 10:3045 (1999); Murata et al., 1059(1997); Murata et al., 1049 (2006); WO2003/046150; WO2007/133855; andKoeller et al., Nature Biotechnology 18:835-841 (2000). However, forefficient transglycosylation by enzymes, a starting population having auniform glycosylation profile is still highly desirable (cf., e.g., thesingle GlcNAc population used by Witte et al., J. Am. Chem. Soc.119:(9)2114-2118, 1997).

A special situation presents itself in crystallization studies ofglycoproteins. Here, N-glycosylation often poses a problem. Indeed, whenattempting to crystallize a glycoprotein, the results can be improvedwhen using de-N-glycosylated forms of the target protein. However,mutation of the glycosylation site is mostly not an option, sinceN-glycosylation is needed for protein folding and quality control. Atpresent, endoH-type endoglycosidases are often used for thepost-purification deglycosylation of high-mannose type glycoproteins.This approach is successful in many cases but contributes to thecomplexity of the downstream processing of these often labile proteins.Therefore, it would be advantageous to be able to eliminate downstreamprocessing steps and still obtain a population that can be used forcrystallization purposes. A similar situation is observed inglycoproteins that are produced in cells that modify them withimmunogenic glycans.

Despite the many advantages of the enzymatic synthesis methods set forthabove, in some cases, deficiencies remain. The preparation of properlyglycosylated glycopeptides is an exemplary situation in which additionaleffort is required and effort is being directed to improving both thesynthesis of glycopeptides and methods of remodeling biologically orchemically produced glycopeptides that are not properly glycosylated.Thus, there is a need to have a cell system or synthesis methodproviding homogeneous (uniform) glycosylation on a population ofglycoproteins, either already with a correct glycoprofile or as astarting point for subsequent transglycosylation. Alternatively, itwould be advantageous to have a cell system or synthesis methodproviding the possibility of easier isolation of the correctly modifiedpopulation of glycoproteins from a mixed population of glycoproteins.Particularly also for yeast, it would be advantageous to be able toeliminate downstream processing steps, while still being able to easilyseparate the desired (complex type) glycoproteins from the undesired,possibly immunogenic glycoforms; or even to obtain yeast cells that nolonger produce immunogenic glycoproteins.

DISCLOSURE

Provided are systems and methods for obtaining desired glycosylationprofiles of a glycoprotein that are economical in both cost and time.The methods can be cheaper and faster than existing methods becausethere is no need for adding an enzyme to the produced glycoprotein inorder to remove the undesired glycosylation products. Correctglycosylation of the glycoprotein (or an essentially homogeneousglycosylated population of an intermediate glycoform of theglycoprotein) is achieved by producing the glycoprotein and anendoglucosaminidase enzyme in the same cellular system. Alsoparticularly envisaged are glyco-engineered yeast cells and methods withthese cells that allow easier isolation of the desired glycoforms of theglycoprotein by selectively deglycosylating the undesired glycoforms,thus allowing easier separation of different glycoforms of secretedproteins. Alternatively, the yeast cells only secrete glycoproteins withthe desired (typically complex) glycosylation pattern.

Thus, according to a first aspect, eukaryotic cells are provided with afirst exogenous nucleic acid sequence (“polynucleotide”) encoding anendoglucosaminidase enzyme and a second exogenous nucleic acid sequenceencoding a glycoprotein. According to particular embodiments, theeukaryotic cells do not express an endogenous endoglucosaminidaseenzyme. According to alternative particular embodiments, the eukaryoticcells do not express an enzyme with functional endoglucosaminidaseactivity other than the endoglucosaminidase enzyme encoded by the firstexogenous nucleic acid sequence.

That such a strategy works is particularly surprising, since a toostrong deglycosylation of cell membrane components by the exogenousendoglucosaminidase would be expected to lead to cell membraneweakening, ultimately leading to cell lysis. This is particularly truefor deglycosylation of mannoproteins of the yeast cell wall.

Eukaryotic cells can be of any eukaryotic organism, but in particular,yeast, plant, mammalian and insect cells are envisaged. According tofurther particular embodiments, the yeast is a Saccharomyces species, aHansenula species, a Yarrowia species or a Pichia species. According toa specific embodiment, the eukaryotic cells are Pichia cells. Accordingto an alternative specific embodiment, the mammalian cells are HEK293cells. According to a very particular embodiment, the eukaryotic cellsare not yeast cells.

According to particular embodiments, the cells possess a third exogenousnucleic acid sequence encoding a glycosyltransferase enzyme. Accordingto specific alternative embodiments, the endoglucosaminidase andglycosyltransferase activity are performed by the same enzyme and thusencoded by the same polynucleotide.

According to very specific embodiments, the eukaryotic cells areglyco-engineered yeast cells, i.e., a yeast cell having inactivatedendogenous glycosylation enzymes and/or comprising at least a thirdexogenous nucleic acid sequence encoding at least one enzyme needed forcomplex glycosylation. Endogenous glycosylation enzymes that could beinactivated include the alpha-1,6-mannosyltransferase Och1p, Alg3p,alpha-1,3-mannosyltransferase of the Mnn1p family,beta-1,2-mannosyltransferases. Enzymes needed for complex glycosylationinclude, but are not limited to: N-acetylglucosaminyl transferase I,N-acetylglucosaminyl transferase II, mannosidase II,galactosyltransferase, fucosyltransferase and sialyltransferase, andenzymes that are involved in donor sugar nucleotide synthesis ortransport. According to particular embodiments, the glyco-engineeredyeast cell may be characterized in that at least one enzyme involved inthe production of high mannose structures (high mannose-type glycans) isnot expressed. Enzymes involved in the production of high mannosestructures typically are mannosyltransferases. In particular,alpha-1,6-mannosyltransferase Och1p, Alg3p,alpha-1,3-mannosyltransferase of the Mnn1p family,beta-1,2-mannosyltransferases may not be expressed.

According to particular embodiments, the endoglucosaminidase enzymeencoded by the first exogenous nucleic acid sequence is amannosyl-glycoprotein endo-beta-N-acetylglucosaminidase, i.e., it hasthe activity of E.C. 3.2.1.96 in the IUBMB nomenclature. According tofurther particular embodiments, the endoglucosaminidase is EndoH orEndoT. According to yet further particular embodiments, theendoglucosaminidase is EndoT.

Provided are efficient and easy-to-implement systems for glycoproteinproduction. Thus, the glycoprotein that is produced by the cell willtypically be easily recovered. It may, for instance, be produced ininclusion bodies, membrane-bound organelles or similar structures in thecell. In particular circumstances, recovery may be achieved by celllysis if the glycoprotein accumulates intracellularly. When cells arepart of an organism that is used for production (e.g., a plant insteadof a plant cell culture), the glycoprotein may be produced in ortransported to specific organs or tissues of the organism from which itcan be recovered (e.g., glands or trichomes). According to particularembodiments, however, the glycoprotein is secreted by the cell. Thistakes away the need for possible refolding or re-activating steps neededwhen the protein is inactive in inclusion bodies. According to furtherspecific embodiments, the endoglucosaminidase is also secreted by thecell.

Although the endoglucosaminidase may be secreted by the cells describedherein, it can be a particular advantage that it remains in the cell.Indeed, this takes away the need for separation of theendoglucosaminidase and the glycoprotein, e.g., when both are secreted.Most particularly, the endoglucosaminidase remains in the cell where itis fully active and, moreover, active at the right place and time.According to a particular embodiment, the endoglucosaminidase isoperably linked to an ER or Golgi localization signal. This ensureslocalization of the endoglucosaminidase to the ER or Golgi,respectively, where it remains in the cell and is in the correctintracellular location to modify the glycosylation of the glycoprotein.Such localization signals are known in the art and may be derived fromproteins that are normally localized in the ER or Golgi for theirfunction. According to particular embodiments, the ER or Golgilocalization signal is from a protein selected from the group of Ste13p,GM2-synthase, and α-2,6-sialyltransferase. Of note, in theglyco-engineered yeast cells described herein, the at least one enzymeneeded for complex glycosylation is/are also localized in the ER orGolgi, to ensure that they successfully modify the glycosylationpathway. This has extensively been described in the art.

The glycosylation status of the produced glycoprotein will depend bothfrom the cellular system used and the specificity of theendoglucosaminidase. In the case of the glyco-engineered yeast cells,this will typically also depend on the enzymes for complex glycosylationpresent in the cells. Moreover, the time and place where these enzymesact is then also important (e.g., which enzyme acts first in theER→Golgi pathway).

Thus, it possible that cells will express solely non-glycosylatedproteins or proteins having only single GlcNAc residues (e.g., in thecase of yeast cells and an endoglucosaminidase capable of hydrolyzinghigh-mannose and hybrid-type glycans). These proteins can serve as thebasis for, e.g., crystallization studies or non-immunogenicglycoproteins. Another (or a further) possibility is that such proteinsare further modified, e.g., by treatment with glycosyltransferases,resulting in proteins with the desired glycan moieties.

Alternatively, cells can be used capable of achieving the desired(typically complex) glycosylations. For instance, yeast can be usedwherein the endoglucosaminidase acts after the enzymes needed forcomplex glycosylation (either intracellularly, e.g., in the trans Golgior trans-Golgi network, or extracellularly). A prerequisite in thisscenario is that the endoglucosaminidase does not hydrolyze the desiredsugar chains on the glycoproteins. Typically, such cells will producetwo populations of glycoproteins: the correctly glycosylated form and anon-glycosylated or single GlcNAc-modified form (obtained, e.g., fromdeglycosylation of glycoproteins with hybrid-type or mannose-type glycanmodifications). Although such mixed population still requires aseparation step before a uniformly glycosylated population is obtained,this separation step is much easier than with traditional productionmethods, as the (e.g., weight) difference between proteins with complexglycosylation and non-glycosylated proteins is much larger than betweendifferently glycosylated proteins.

Alternatively, it is envisaged that the cells produce and/or secreteonly correctly glycosylated proteins, e.g., by recycling thenon-glycosylated proteins. This may, for instance, be achieved byredirecting non-glycosylated proteins to the ER-Golgi machinery, whileglycoproteins with complex glycosylation are secreted. Inglyco-engineered yeast cells, the secretion of correctly glycosylatedproteins may be achieved, e.g., by targeting the endoglucosaminidaseenzyme just before the at least one enzyme for complex glycosylation inthe ER→Golgi pathway, in such a way that all glycoproteins are first (atleast partly) deglycosylated by the endoglucosaminidase, after whichthey are modified by the at least one enzyme for complex glycosylation.Using the latter approach, the produced glycoproteins may havenon-naturally occurring carbohydrate chains, as the endoglucosaminidasetypically will remove the core Man₅GlcNAc₂ structure, or at least partthereof, so that the sugar chain added on the glycoprotein by theenzymes for complex glycosylation will be added on shortened basestructures, such as a single GlcNAc residue. Although not naturallyoccurring, such complex sugar chains often also are non-immunogenic andmay have other desirable properties, such as, e.g., increased stability,longer half-life, etc.

However, it is understood that, especially in cells other than specificglyco-engineered yeast cells described herein, further (complex)glycosylation may also be inhibited, e.g., in order to retain solelysingle GlcNAc-modified proteins. This may have advantages with regard toimmunogenicity or downstream handling (e.g., for crystallization or forproviding a uniform population of glycoproteins). Thus, according to aparticular embodiment, the eukaryotic cells described herein do notcomprise at least one functional enzyme needed for complexglycosylation, such as ER-mannosidase I, Glucosidase I, Glucosidase II,N-acetylglucosaminyl transferase I, mannosidase II, N-acetylglucosaminyltransferase II. Such cells are not capable of complex glycosylation ofglycoproteins. Absence of enzyme activity may be obtained throughgenetic inactivation strategies such as homology-based knockout,insertion mutagenesis, random mutagenesis, or through transcriptionaland/or translational silencing as may be obtained through, for example,siRNA strategies, or through inhibition of the enzyme with chemicalinhibitors (e.g., kifunensine for ER-mannosidase-I, castanospermine forglucosidases, or swainsonine for mannosidase II).

Whereas cells for the production of glycoproteins as described hereinwill typically be provided in the form of a cell culture, this need notnecessarily be the case. Indeed, the cells producing the glycoproteinsmay be part of an organism, e.g., a transgenic animal or plant.According to a particular embodiment, plants comprising the cellscontaining a glycoprotein and an endoglucosaminidase, as described inthe application, are also envisaged.

Also provided in the application are methods using the cells describedherein. Particularly, methods are provided for producing singleGlcNac-modified glycoproteins in a eukaryotic cell, comprising the stepsof:

-   -   providing a eukaryotic cell comprising a first exogenous nucleic        acid sequence encoding an endoglucosaminidase enzyme and a        second exogenous nucleic acid sequence encoding a glycoprotein        in conditions suitable for expressing the endoglucosaminidase        enzyme and the glycoprotein; and    -   recovering the glycoprotein after it has been intracellularly or        extracellularly contacted with the endoglucosaminidase.

The glycoproteins with a single GlcNAc residue may be the only glycoformof the glycoprotein produced by the cell, i.e., a uniformglycopopulation is produced. Alternatively, several glycoforms of theglycoprotein may be produced, but these typically can be easilyseparated (e.g., proteins with complex glycosylation as well as proteinswith single GlcNAc residues). Typically, these several glycoforms willbe limited in number (e.g., two glycoforms), as a more or less uniformglycoprofile is desirable. According to particular embodiments, theeukaryotic cells used in the methods described herein are not capable ofcomplex glycosylation of glycoproteins.

Particularly for the specific glyco-engineered yeast cells describedherein, methods are provided for producing proteins in aglyco-engineered yeast cell while depleting proteins with highmannose-type glycosylation and/or hybrid-type glycosylation, comprisingthe steps of:

-   -   providing a glyco-engineered yeast cell comprising a first        exogenous nucleic acid sequence encoding an endoglucosaminidase        enzyme, a second exogenous nucleic acid sequence encoding a        glycoprotein, and at least a third exogenous nucleic acid        sequence encoding at least one enzyme needed for complex        glycosylation, selected from the group consisting of        mannosidases and glycosyltransferases other than        mannosyltransferases and phosphomannosyltransferases, in        conditions suitable for expressing these enzymes and the        glycoprotein; and    -   recovering the glycoprotein after it has been intracellularly        contacted with the at least one enzyme needed for complex        glycosylation and intracellularly or extracellularly contacted        with the endoglucosaminidase.

Depleting proteins with high mannose-type glycosylation and/orhybrid-type glycosylation in these yeast cells may result in yeast cellsproducing glycoproteins as a uniform and homogeneous, typically complex,glycopopulation. Alternatively, several glycoforms of the glycoproteinmay be produced, but these typically can be easily separated as noglycoproteins with sugar chains of comparable size to the complexglycans are produced. An example of mixed glycoforms that are producedare proteins with complex glycosylation as well as proteins with singleGlcNAc residues.

For all methods, it is true that to ensure that the contact with theendoglucosaminidase occurs under optimal circumstances (i.e., to ensureoptimal activity of the endoglucosaminidase on the glycoprotein), themethods may be optimized to suit the desired purpose. For instance, whenthe contact occurs intracellularly, the endoglucosaminidase may betargeted to the (right place in the) Golgi or ER where it exerts itsfunction on the glycoprotein. According to a particular embodiment, theintracellular contact occurs in the Golgi or ER.

Of note, for the specific glyco-engineered yeast cells, the at least oneenzyme needed for complex glycosylation will typically also be localizedin (i.e., targeted to) the Golgi or ER, as these are the organelleswhere the process of glycosylation typically occurs. According tofurther particular embodiments, the respective targeting signals of theendoglucosaminidase and the enzyme needed for complex glycosylation arechosen in such a way that the enzymes are targeted to differentfunctional regions (endoplasmic reticulum, cis-Golgi network, cis-Golgi,medial Golgi, trans-Golgi, trans-Golgi network) so that they actsequentially. According to yet further particular embodiments, theenzymes are targeted in such a way that they act immediately after eachother, e.g., they may be targeted to adjacent compartments in the Golgiapparatus.

When the enzymes are targeted to act sequentially, the glycoprotein mayfirst be contacted with the at least one enzyme needed for complexglycosylation or alternatively with the endoglucosaminidase. Accordingto particular embodiments, the intracellular contact with theendoglucosaminidase occurs in the Golgi or ER, after contact with the atleast one enzyme needed for complex glycosylation.

Although the endoglucosaminidase, like the at least one enzyme neededfor complex glycosylation, may be retained in the cell (and, inparticular, within the ER→Golgi region where glycosylation occurs), incase the glycoprotein is secreted, it is also possible for theendoglucosaminidase to be secreted and the contact between glycoproteinmay happen extracellularly. In this case, the (intracellular) contactwith the at least one enzyme needed for complex glycosylation takesplace before the (extracellular) contact with the endoglucosaminidase.

For all of the cells described herein, methods may imply that bothproteins may also be secreted and the contact may happenextracellularly. Depending on the cells and endoglucosaminidase that areused, however, the optimal growth conditions for the cells (e.g., pH,temperature, nature of medium) may differ from the optimal conditionsfor enzymatic activity. Thus, the medium where the extracellular contactbetween the glycoprotein and the endoglucosaminidase takes place may beadjusted for optimal enzymatic activity of the endoglucosaminidase.According to a particular embodiment, the conditions of the mediumwherein the extracellular contact takes place are adjusted for optimalenzymatic endoglucosaminidase activity. According to a furtherparticular embodiment, the pH of the medium wherein the extracellularcontact takes place is adjusted for optimal enzymaticendoglucosaminidase activity. Typically, this may be done by a pH shiftof the medium after the cells have been allowed to produce and secreteboth glycoproteins and endoglucosaminidases. In general, such pH shiftwill be a downshift, as endoglucosaminidases usually are physiologicallyactive in an acidic environment (e.g., the Golgi apparatus within acell). Alternatively, the cells may be grown in a medium with a pH thatis both permissive for growth and enzymatic activity, so that no pHshift is necessary. According to another particular embodiment, thetemperature of the medium is adjusted for optimal enzymatic activity.According to yet another particular embodiment, the nature of the medium(e.g., salt or ion content and/or concentration) is adjusted for optimalenzymatic activity.

According to a particular aspect, the protein modified with the singleGlcNAc residues is not the end-point. Methods according to this aspectwill include at least one additional glycosylation step. According tothis embodiment, before the final recovery of the glycoprotein, themethods further involve a step of contacting the enzyme with aglycosyltransferase after it has been intracellularly or extracellularlycontacted with the endoglucosaminidase. Optionally, this contacting witha glycosyltransferase may occur in the presence of specific glycosyldonors (e.g., sugar nucleotide donors) to ensure efficient and correctglycosylation. This will especially be the case when the glycosylationtakes place extracellularly.

If the transglycosylation step takes place intracellularly, it will beunderstood by the skilled person that, when both the endoglucosaminidaseenzyme and the glycosyltransferase enzyme are targeted to the ER orGolgi, it is ensured that the glycosyltransferase activity occurs afterthe endoglucosaminidase activity. Typically, this may be ensured bytargeting both enzymes to different compartments of the ER or Golgi, asthere is a fixed order in which proteins follow the ER→Golgi route. Inthe event both enzymes are targeted to the same compartment, or thatboth activities are performed by the same enzyme, it typically will beensured that the protein after the transglycosylation step is no longerrecognized as substrate for the endoglucosaminidase enzyme. Thus,separation of the enzymatic activities in time may involve spatialseparation and/or a different substrate specificity. According to aparticular embodiment, both the endoglucosaminidase and theglycosyltransferase are produced by the same cell, but only theglycosyltransferase is secreted, to ensure transglycosylation takesplace after the endoglucosaminidase activity.

Depending on how the method is performed, the glycosyltransferase enzymemay be added extracellularly (i.e., is not produced by the same cells),is also produced and secreted by the cells producing the glycoproteinand endoglucosaminidase, or is also produced by the cells and retainedwithin the ER or Golgi apparatus. The glycosyltransferase may be encodedby an exogenous sequence, or may be an enzyme that is endogenous in thecells having a first exogenous nucleic acid sequence encoding anendoglucosaminidase enzyme and a second exogenous nucleic acid sequenceencoding a glycoprotein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Released N-glycans from RNase B after in vitro treatment withEndoH (second panel), different purified forms of EndoT (as indicated)(panels 3-5) and PNGase F (panel 6).

FIG. 2: Proteolytic activity of different EndoT fusion constructs. Lane1: medium of hIFNβ strain; lane 2: medium of hIFNβ strain transformedwith fusion construct 2; lane 3: medium of hIFNβ strain transformed withfusion construct 1; lane 4: no medium added.

FIG. 3: Glycan profiles of a GS115 yeast strain overexpressing IFNβ(panel 2), different clones also expressing EndoT (panels 3-5), yeasttreated with RNase B (panel 6), and the strains of panels 2 and 3 withlowering pH to 5 in the induced medium (panels 7-8).

FIGS. 4A and 4B: FIG. 4A, Glycan profiles of wild-type GS115 or FIG. 4B,Man5-glyco-engineered Pichia strains not expressing EndoT (panels 2) orsoluble overexpressing different EndoT forms (N-terminal truncated,C-terminal truncated, both N- and C-terminal truncated or full size).

FIGS. 5A through 5F: Glycan profiles of different glyco-engineered yeaststrains producing GM-CSF as glycoprotein. FIG. 5A, Gal2GlNAc2Man3strain; FIG. 5B, GalGlcNacMan3 strain; FIG. 5C, GalGlcNAcMan5 strain;FIG. 5D, GlcNAcMan5 strain; FIG. 5E, Man5 strain; and FIG. 5F, WT GS115strain. Panel 2, PNGase F treatments; Panel 3, EndoH treatment; Panel 4,EndoT treatment; Panel 5, EndoH treatment followed by PNGase Ftreatment; Panel 6, EndoT treatment followed by PNGase F treatment;Panel 7, RNase B treatment. In panel 5 of FIG. 5F a contaminatingpolymer is present.

FIG. 6: Western blot for Flt3 expression in Hek293 cells, detection withpenta-His primary antibody. Lane 1: positive control; lanes 2-6: 48hours post-transfection; lanes 7-11: 72 hours post-transfection; lanes 2and 7: supernatant from pCAGGS transfected cells (negative control);lanes 3 and 8: supernatant from pCAGGS-hGalNAcT-endoT transfected cells(i.e., with endoT fused to human GM2-synthase targeting domain); lanes 4and 9: supernatant from pCAGGS-hGalNAcT-endoT-myc transfected cells(with myc-tag); lanes 5 and 10: supernatant from pCAGGS-hST-endoTtransfected cells (i.e., with EndoT fused to humanβ-galactoside-α-2,6-sialyltransferase targeting domain); lanes 6 and 11:supernatant from pCAGGS-hST-endoT-myc transfected cells. After threedays, fully glycosylated Flt3 is only detectable in the negative control(lane 7), indicating that EndoT is functional in all EndoT transfectedcells.

DETAILED DESCRIPTION Definitions

The disclosure will be described with respect to particular embodimentsand with reference to certain drawings but the disclosure is not limitedthereto; only by the claims. Any reference signs in the claims shall notbe construed as limiting the scope. The drawings described are onlyschematic and are non-limiting. In the drawings, the size of some of theelements may be exaggerated and not drawn on scale for illustrativepurposes. Where the term “comprising” is used in the present disclosure,it does not exclude other elements or steps. Where an indefinite ordefinite article is used when referring to a singular noun, e.g., “a,”“an,” or “the,” this includes a plural of that noun unless somethingelse is specifically stated.

Furthermore, the terms “first,” “second,” “third” and the like in thedescription and in the claims, are used for distinguishing betweensimilar elements and not necessarily for describing a sequential orchronological order. It is to be understood that the terms so used areinterchangeable under appropriate circumstances and that the embodimentsof the disclosure described herein are capable of operation in othersequences than described or illustrated herein.

The following terms or definitions are provided solely to aid in theunderstanding of the disclosure. Unless specifically defined herein, allterms used herein have the same meaning as they would to one skilled inthe art of the present disclosure. Practitioners are particularlydirected to Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor Press, Plainsview, N.Y. (1989); andAusubel et al., Current Protocols in Molecular Biology (Supplement 47),John Wiley & Sons, New York (1999), for definitions and terms of theart. The definitions provided herein should not be construed to have ascope less than understood by a person of ordinary skill in the art.

“Glyco-engineered yeast cells,” as used in the application, are yeastcells that express at least one exogenous nucleic acid sequence encodingan enzyme needed for complex glycosylation that is not expressed in thewild-type yeast, and/or that do not express at least one enzyme involvedin the production of high-mannose type structures that is normallyexpressed in the wild-type yeast.

An “endoglucosaminidase,” as used herein, refers to enzymes thathydrolyze the bond between the anomeric carbon of a non-terminalbeta-linked N-acetylglucosamine residue in an oligosaccharide of aglycoprotein or a glycolipid, and its aglycon, thereby releasing mono-or oligosaccharides from glycoproteins or glycolipids or sugar polymers.Endoglucosaminidases are a subset of the glycosidases, and may or maynot have other enzymatic activities (such as, e.g., glycosyltransferaseactivity). A particular class of endoglucosaminidases is formed by theendo-β-N-acetylglucosaminidases or mannosyl-glycoproteinendo-β-N-acetylglucosaminidases, indicated as EC 3.2.1.96 in theInternational Union of Biochemistry and Molecular Biology (IUBMB)nomenclature. This particular class of enzymes are capable of catalyzingthe endohydrolysis of the N,N′-diacetylchitobiosyl unit in high-mannoseglycopeptides and glycoproteins containing the -[Man(GlcNAc)₂]Asn-structure. One N-acetyl-D-glucosamine (GlcNAc) residue remains attachedto the protein; the rest of the oligosaccharide is released intact.Thus, the result is a single GlcNAc-modified glycoprotein. Of note, theremaining GlcNAc residue may be either unmodified or still be modifiedwith other sugar residues in other positions than that of the hydrolyzedbond, for instance, the GlcNAc residue may carry a fucose on position 3or 6. Nevertheless, glycoproteins with a modified GlcNAc residue willstill be referred to as single GlcNAc-modified proteins, as there is nosecond sugar residue on position 4 of the GlcNAc residue (i.e., there isno typical sugar chain). A particular advantage of endoglucosaminidasesas compared to exoglycosidases is that they allow discrimination betweenN-linked and O-linked glycans and between classes of glycans. Anon-limiting list of endoglucosaminidases is provided in theapplication.

Particularly with regard to the glyco-engineered yeast cells, an “enzymeneeded for complex glycosylation,” as used herein, refers to any enzymenot naturally occurring in the host yeast cell that may be involved inthe synthesis of complex glycans as found in higher eukaryotes, inparticular, as found in mammals, more in particular, as found in humans.Most particularly, such enzymes are enzymes that remove mannose residuesfrom the sugar chain (i.e., mannosidases) or glycosyltransferases, inparticular, glycosyltransferases other than mannosyltransferases (i.e.,glycosyltransferases that transfer monosaccharides that are not found inhigh-mannose glycans) and/or phosphomannosyltransferases.

A “glycosyltransferase” as used in the application is any of a group ofenzymes that catalyze the transfer of glycosyl groups in biochemicalreactions, in particular, glycosyl transfer to asparagine-linked sugarresidues to give N-linked glycoproteins. Glycosyltransferases fall underEC 2.4 in the IUBMB nomenclature, a particular class ofglycosyltransferases are hexosyltransferases (EC 2.4.1). Among the widevariety of these post-translational enzymes that process peptides intoglycoproteins are enzymes such as, but not limited to,N-acetylglucosaminyl transferases, N-acetylgalactosaminyltransferases,sialyltransferases, fucosyltransferases, galactosyltransferases, andmannosyltransferases.

Note that exogenous mannosyltransferases are excluded for specificembodiments of glyco-engineered yeast cells described in theapplication. “Mannosyltransferases” as used in the application refers toenzymes that catalyze the transfer of a mannosyl group to an acceptormolecule, typically another carbohydrate, in the Golgi apparatus.Mannosyltransferases are typically endogenous enzymes in yeast andinvolved in the synthesis of high-mannose type glycans.

Of note, an enzyme may possess both endoglucosaminidase andglycosyltransferase activity. Although it may be possible to use oneenzyme to exert these two activities, typically, the enzymes used willfulfill only one function. Thus, it is envisaged to use enzymes thathave been modified or mutated to make sure they perform only onefunction, or that have been modified or mutated to ensure they carry outa specific function more efficiently. Such modified enzymes are known inthe art.

“Glycoproteins” as used in the application refers to proteins that, intheir normal physiological context and/or their functional form, containoligosaccharide chains (glycans) covalently attached to theirpolypeptide side-chains. The carbohydrate may be attached to the proteinin a co-translational or post-translational modification. In particular,“glycoproteins” as used herein are proteins that show N-glycosylation intheir physiologically active form. Thus, glycoproteins typically containa sugar chain at least on one asparagine residue. A non-limiting list ofglycoproteins is provided in the specification. The term “glycoproteins”is not intended to refer to the length of the amino acid chain,“glycopeptides” are included within the definition of “glycoproteins.”

The terms “(glyco)protein” and “enzyme” (e.g., endoglucosaminidase,glycosyltransferase, mannosidase, mannosyltransferase) as used in theapplication are also intended to cover functionally active fragments andvariants of the naturally occurring proteins. Indeed, for many (e.g.,therapeutic) proteins, part of the protein may be sufficient to achievean (e.g., therapeutic, enzymatic) effect. The same applies for variants(i.e., proteins in which one or more amino acids have been substitutedwith other amino acids, but which retain functionality or even showimproved functionality), in particular, for variants of the enzymesoptimized for enzymatic activity.

In the context of the application, a glycoprotein refers to the proteinitself; a glycoprotein may be either in its glycosylated ornon-glycosylated form. A “glycosylated” protein is a (glyco)protein thatcarries at least one oligosaccharide chain.

A “sugar chain,” “oligosaccharide chain” or “carbohydrate chain,” asused herein, is a chain of two or more monosaccharides. As aconsequence, a protein carrying only a single monosaccharide (e.g., asingle GlcNAc residue) will usually, unless specified otherwise, not bereferred to as a glycosylated protein, but as a protein that carries amonosaccharide, or a monosaccharide (e.g., GlcNAc)-modified protein.Typical monosaccharides that may be included in an oligosaccharide chainof a glycoprotein include, but are not limited to, glucose (Glu),galactose (Gal), mannose (Man), fucose (Fuc), N-acetylneuraminic acid(NeuAc) or another sialic acid, N-acetylglucosamine (GlcNAc),N-acetylgalactosamine (GalNAc), xylose (Xyl) and derivatives thereof(e.g., phosphoderivatives). Sugar chains may be branched or not, and maycomprise one or more types of oligosaccharide. In general, sugar chainsin N-linked glycosylation may be divided in three types: high-mannose,complex and hybrid type glycosylation. These terms are well known to theskilled person and defined in the literature. Briefly, high-mannose typeglycosylation typically refers to oligosaccharide chains comprising twoN-acetylglucosamines with (possibly many) mannose and/ormannosylphosphate residues (but typically no other monosaccharides).

Complex glycosylation typically refers to structures with typically one,two or more (e.g., up to six) outer branches with a sialyllactosaminesequence, most often linked to an inner core structure Man₃GlcNAc₂. Forinstance, a complex N-glycan may have at least one branch, or at leasttwo, of alternating GlcNAc and galactose (Gal) residues that mayterminate in a variety of oligosaccharides but typically will notterminate with a mannose residue.

Hybrid type glycosylation covers the intermediate forms, i.e., thoseglycosylated proteins carrying both terminal mannose and terminalnon-mannose residues in addition to the two N-acetylglucosamineresidues. In contrast to complex glycosylation, at least one branch ofhybrid type glycosylation structures ends in a mannose residue.

Although this classification is most often used to describe naturallyoccurring glycans on proteins, it is evident that synthetic and/ornon-naturally occurring sugars can also be classified this way, even iftheir structures diverge from the classical example. For instance, asugar chain consisting of a single branch of a galactose and a sialicacid residue linked to a single GlcNAc would be a complex sugar, eventhough it lacks the inner core Man₃GlcNAc₂.

An “ER localization signal” or a “Golgi localization signal” is amolecule, typically a peptide that directs localization of thepolypeptide or protein to which it is conjugated to the ER or Golgiapparatus, respectively. Localization thus also implies retention in theER or Golgi apparatus, respectively. Typically, these localization (orretention) sequences are peptide sequences derived from (pre)proteinsthat are situated in the ER or Golgi when functionally active as amature protein.

The disclosure aims to provide cells producing glycoproteins with analtered glycosylation pattern, in particular, a more homogeneousglycosylation pattern, that makes them more amenable for further use,e.g., therapeutic use, or use in crystallization studies. This isachieved, according to a first aspect, by providing eukaryotic cellswith a first exogenous nucleic acid sequence encoding anendoglucosaminidase enzyme and a second exogenous nucleic acid sequenceencoding a glycoprotein. The nature of the glycoprotein is not critical,but glycoproteins will typically be proteins relevant for medicineand/or industry for which correct N-glycosylation is important for theirfunction. Non-limiting examples include many hormones, growth factors,cytokines and their corresponding receptors, such asfollicle-stimulating hormone (FSH), luteinizing hormone (LH),thyroid-stimulating hormone (TSH), epidermal growth factor (EGF), humanepidermal growth factor receptor-2 (HER-2), fibroblast growthfactor-alpha (FGF-α), fibroblast growth factor-beta (FGF-β),transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), platelet-derived growth factor (PDGF), insulin-likegrowth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), nervegrowth factor (NGF), nerve growth factor-beta (NGF-β); receptors of theaforementioned, growth hormones (e.g., human growth hormone, bovinegrowth hormone); insulin (e.g., insulin A chain and insulin B chain),proinsulin; erythropoietin (EPO); colony-stimulating factors (e.g.,granulocyte colony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), macrophage colony-stimulating factor(M-CSF)); interleukins (e.g., IL-1 through IL-12); vascular endothelialgrowth factor (VEGF) and its receptor (VEGF-R); interferons (e.g.,IFN-α, β, or γ); tumor necrosis factor (e.g., TNF-α and TNF-β) and theirreceptors, TNFR-1 and TNFR-2; thrombopoietin (TPO); thrombin; brainnatriuretic peptide (BNP); clotting factors (e.g., Factor VIII, FactorIX, von Willebrands factor, and the like); anti-clotting factors; tissueplasminogen activator (TPA), e.g., urokinase or human urine or tissuetype TPA; calcitonin; CD proteins (e.g., CD3, CD4, CD8, CD28, CD19,etc.); CTLA proteins (e.g., CTLA4); T-cell and B-cell receptor proteins;bone morphogenic proteins (BMPs, e.g., BMP-1, BMP-2, BMP-3, etc.);neurotrophic factors, e.g., bone-derived neurotrophic factor (BDNF);neurotrophins, e.g., 3-6; renin; rheumatoid factor; RANTES; albumin;relaxin; macrophage inhibitory protein (e.g., MIP-1, MIP-2); viralproteins or antigens; surface membrane proteins; ion channel proteins;enzymes; alkaline phosphatase; lectins; regulatory proteins; antibodies;immunomodulatory proteins, (e.g., HLA, MHC, the B7 family); homingreceptors; transport proteins; superoxide dismutase (SOD); G-proteincoupled receptor proteins (GPCRs); neuromodulatory proteins; Alzheimer'sDisease associated proteins and peptides, (e.g., A-beta), and others asknown in the art, including fusion or chimeric proteins of the above.Fragments or portions, or mutants, variants, or analogues of any of theaforementioned proteins and polypeptides are also included among thesuitable proteins, polypeptides and peptides that can be produced by thecells and methods presented herein.

The nature of the endoglucosaminidase will depend on the desiredglycopopulation of the glycoproteins. For instance, endoglucosaminidasesmay be selected for their substrate specificity. Someendoglucosaminidases, e.g., EndoH and EndoT, hydrolyze high-mannose typesugar chains and hybrid type sugars, but leave complex carbohydratestructures intact. Such enzymes are ideal, e.g., for obtaining singleGlcNAc-modified glycoproteins from cells incapable of complexglycosylation, or for removing contaminating high-mannose and/or hybridtype sugars in cells producing complex glycosylated proteins as well asother glycoforms (such as most glyco-engineered yeast strains).According to particular embodiments, the endoglucosaminidase hydrolyzeshigh mannose-type sugar chains and hybrid-type glycans, but notcomplex-type glycans.

Endoglucosaminidases may also have substrate specificity with regard tothe glycoprotein (instead of only the sugar chain), someendoglucosaminidases are, e.g., more successful in hydrolyzing sugarchains from (particularly compactly folded) proteins than otherendoglucosaminidases (e.g., EndoT); others may (also) be particularlysuccessful in hydrolyzing sugar chains from glycopeptides ornot-compactly folded proteins (e.g., EndoH, EndoT). Importantly, as thistypically has to do with access to or availability of the substraterather than with the specificity of the endoglucosaminidase, this doesnot exclude the use of certain enzymes for specific proteins, but someendoglucosaminidases may require more time to complete the hydrolysis ofall N-linked sugar structures.

The choice of endoglucosaminidases may also depend on the resultingproduct(s). For instance, when different glycopopulations are secreted(e.g., complex-type glycosylated proteins that are not hydrolyzed andother types that are hydrolyzed), it may be important that the resultingproteins can be easily separated. As another example, when furthertransglycosylation is envisaged, endoglucosaminidases leaving singleGlcNAc-modified proteins (e.g., EndoH, EndoT) are particularlyenvisaged, as the single GlcNAc residue on the protein offers a suitablesubstrate for the glycosyltransferase to attach the sugar modification.This is a significant advantage of the eukaryotic cells described hereinas compared to bacterial expression systems, as the bacteria cannotproduce single GlcNAc-modified glycoproteins, which makes it much moredifficult to use proteins produced in bacteria as starting point fortransglycosylation. Alternatively, single GlcNAc-modified proteins canbe used in crystallization studies, although this is also true fornon-glycosylated proteins. Thus, endoglucosaminidases removing the wholesugar chain without leaving a monosaccharide on the protein (such aspeptide-N-glycosidase F) may be envisaged when using the producedglycoproteins for crystallization. Another consideration may be thepresence or absence of other enzymatic activities, such asglycosyltransferase activity. EndoA, EndoBH and EndoM, for instance, areknown to possess such glycosyltransferase activity, and it may for someembodiments be desirable to work with mutants that no longer possessthis activity.

A particular class of endoglucosaminidases is formed by themannosyl-glycoprotein endo-β-N-acetylglucosaminidases, indicated as EC3.2.1.96 in the IUBMB nomenclature. These enzymes can remove sugarchains while leaving one GlcNAc residue on the protein. Examples ofthese include, but are not limited to, EndoA, EndoBH, EndoCE, EndoD,EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoT (see, also WO2006/050584),AcmA, and ENGase. Other examples are known to the skilled person andcan, for instance, be found on the World Wide Web at cazy.org, inparticular, under the Glycoside Hydrolase Families 85 and 18.Particularly envisaged is the use of the EndoT enzyme from Hypocreajecorina (formerly known as Trichoderma reesei) that is described inWO2006/050584 (see, e.g., SEQ ID NOS:9-12 therein).

According to particular embodiments, the eukaryotic cells do not expressan endogenous endoglucosaminidase enzyme, in particular, nomannosyl-glycoprotein endo-β-N-acetylglucosaminidase. According toalternative particular embodiments, the eukaryotic cells do not expressan enzyme with functional endoglucosaminidase activity other than theendoglucosaminidase enzyme encoded by the first exogenous nucleic acidsequence. That is, they may, for instance, express anotherendoglucosaminidase, but an endoglucosaminidase that is modified to nolonger have its hydrolase activity (but, e.g., only itsglycosyltransferase activity, so that it can function in the synthesisof complex glycosylation structures).

The eukaryotic cells as described herein may produce uniformly, singleGlcNAc-modified glycoproteins that are ready to use (e.g., forcrystallization studies), or that may be used as a starting point forfurther glycomodification reactions, e.g., by glycosyltransferases.Alternatively, the eukaryotic cells may produce two populations ofeasily separable, differentially glycosylated glycoproteins, onepopulation of which is typically single GlcNAc-modified. The other willin such case typically have a complex glycosylation pattern, althoughthis is not strictly required.

Glycosyltransferases have been used to modify the oligosaccharidestructures on glycopeptides, and have been shown to be very effectivefor producing specific products with good stereochemical andregiochemical control. Glycosyltransferases may be used to prepareoligosaccharides and to modify terminal N- and O-linked carbohydratestructures on glycopeptides produced in eukaryotic cells. For example,the terminal oligosaccharides may be completely sialylated and/orfucosylated to create sugar structures that improve glycoprotein (orglycopeptides) pharmacodynamics and a variety of other biologicalproperties, such as, e.g., immunogenicity. Such glycosyltransferases maybe used in natural or synthetic pathways, for instance,fucosyltransferases have been used in synthetic pathways to transfer afucose unit from guanosine-5′-diphosphofucose to a specific hydroxyl ofa saccharide acceptor (Ichikawa et al., J. Am. Chem. Soc. 114:9283-9298(1992)).

Under appropriate conditions, both exoglycosidases and endoglycosidaseshave been shown to possess glycosyl transferase activity. Methods basedon the use of endoglycosidases have the advantage that anoligosaccharide, rather than a monosaccharide, is transferred. The aboveenzymes can be utilized in the generation of carbohydrates (that are,e.g., to be conjugated to glycoproteins) as well as glycosylatedglycoproteins themselves. For examples of how glycosyltransferases maybe used in the further processing of, e.g., single GlcNAc-modifiedglycoproteins, see, e.g., Takegawa JBC 3094, Koeller et al., 835, Nat.Biotech. 2000; WO03/046150, and WO07/133855.

However, instead of delivering the intermediary glycoprotein productthat is to be used in further transglycosylation steps with aglycosyltransferase that needs to be added, it is also envisaged thatthe cells described herein may themselves produce theglycosyltransferase(s). Indeed, it is envisaged that theglycosyltransferase(s) of the cells perform a glycosylation reaction onthe glycoproteins, either within the cells or in the extracellularenvironment, thereby yielding a uniform population of glycoproteins withthe desired (typically complex) glycosylation profile.

Thus, according to particular embodiments, the cells possess a thirdexogenous nucleic acid sequence encoding a glycosyltransferase enzyme.According to specific alternative embodiments, the endoglucosaminidaseand glycosyltransferase activity are performed by the same enzyme. Thismay be because there is only one enzyme and both activities are thusencoded by the same sequence (although it is also possible that theenzyme sequence is identical, but the localization or secretion sequencediffers). Alternatively, it is envisaged that two versions of the sameenzyme are expressed in the cell (e.g., EndoT, EndoM), one that hasendoglucosaminidase activity but (preferably) no glycosyltransferaseactivity, and one that has only glycosyltransferase activity. If anenzyme is used that still has both activities, it is important tocontrol (spatiotemporal) access to its substrate, in order to avoidinterference of the two enzymatic activities. For instance, when theenzyme and glycoprotein are secreted, the endoglucosaminidase activitymay be activated first (e.g., by adapting pH), after which substratesfor transglycosylation can be added to the medium. Even so, it should beensured that the endoglucosaminidase is not able to hydrolyze theglycoprotein after it has been modified with a sugar chain by theglycosyltransferase activity.

According to particular embodiments, however, the glycosyltransferase isnot encoded by the same sequence as the endoglucosaminidase. Accordingto further particular embodiments, one or more glycosyltransferasesdifferent from the endoglucosaminidases are used. Examples include, butare not limited to, sialyltransferases such as α-sialyltransferases,galactosyltransferases such as β-1,4-galactosyltransferase, andfucosyltransferases.

According to alternative, but not necessarily exclusive, particularembodiments, the cells are glyco-engineered yeast cells, i.e., yeastcells that also possess at least a third exogenous nucleic acid sequenceencoding at least one enzyme needed for complex glycosylation, and/orare deficient in the activity of at least one endogenousglycosyltransferase. According to particular embodiments, the enzymeneeded for complex glycosylation is a mannosidase or aglycosyltransferase other than a mannosyltransferase. According tofurther particular embodiments, the at least one enzyme needed forcomplex glycosylation is selected from the group consisting ofN-acetylglucosaminyl transferase I, N-acetylglucosaminyl transferase II,mannosidase II, galactosyltransferase, and sialyltransferase.

According to particular embodiments, the glyco-engineered yeast cell maybe characterized in that at least one enzyme involved in the productionof high mannose structures (high mannose-type glycans) is not expressed(or is not functionally active in the cell). According to furtherparticular embodiments, at least one mannosyltransferase is notexpressed in the glyco-engineered yeast cell. Typically, themannosyltransferase that is not expressed in the glyco-engineered yeastcell is expressed in the wild-type counterpart of the yeast cell.According to yet further particular embodiments, the mannosyltransferaseis a α-1,2-mannosyltransferase, α-1,3-mannosyltransferase,α-1,6-mannosyltransferase, or β-1,4-mannosyltransferase. These proteinsoften have specific names in yeast (e.g., Alg, Och, Mnn), but theiractivities are well known in the art. Alternatively or additionally, atleast one mannosylphosphate transferase is not functionally active inthe glyco-engineered yeast cell.

In the eukaryotic cells described herein, the glycosyltransferase may,just like the endoglucosaminidase, be secreted or be retained in thecell, in particular, targeted to the ER or Golgi. In the latter case, itwill typically be targeted to a later stage of the ER→Golgi assemblypathway for glycosylated proteins, to ensure that the proteins are(partly) deglycosylated by the endoglucosaminidase first, after whichthey are subject to transglycosylation by the glycosyltransferase. Thisway, depending on the combinations of endoglucosaminidase(s) andglycosyltransferase(s), naturally occurring as well as synthetic glycanscan be added to the glycoproteins.

Eukaryotic cells can be of any eukaryotic organism, but, in particularembodiments, yeast, plant, mammalian and insect cells are envisaged. Thenature of the cells used will typically depend on the desiredglycosylation properties and/or the ease and cost of producing theglycoprotein. Mammalian cells may, for instance, be used for achievingcomplex glycosylation and avoiding problems with immunogenicity, but itmay not be cost-effective to produce proteins in mammalian cell systems.Plant and insect cells, as well as yeast, typically achieve highproduction levels and are more cost-effective, but additionalmodifications may be needed to mimic the complex glycosylation patternsof mammalian proteins, or to reduce problems with immunogenicity.Eukaryotic cell lines for protein production are well known in the art,including cell lines with modified glycosylation pathways. Nonlimitingexamples of animal or mammalian host cells suitable for harboring,expressing, and producing proteins for subsequent isolation and/orpurification include Chinese hamster ovary cells (CHO), such as CHO-K1(ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet.,12:555-556; and Kolkekar et al., 1997, Biochemistry, 36:10901-10909),CHO-K1 Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR,Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B(GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR,Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC 92052129 (CAMR,Salisbury, Wiltshire, UK), dihydrofolate reductase negative CHO cells(CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA,77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidneyCV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); humanembryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cellssubcloned for growth in suspension culture, Graham et al., 1977, J. Gen.Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkeykidney cells (CV1, ATCC CCL-70); African green monkey kidney cells(VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4,Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells(HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lungcells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mousemammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells(BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals N. Y. Acad.Sci., 383:44-68); MCR 5 cells; FS4 cells. Exemplary non-mammalian celllines include, but are not limited to, Sf9 cells, baculovirus-insectcell systems (e.g., review Jarvis, Virology Volume 310, Issue 1, 25 May2003, pages 1-7), plant cells such as tobacco cells, tomato cells, maizecells, algae cells, or yeasts such as Saccharomyces species, Hansenulaspecies, Yarrowia species or Pichia species. According to particularembodiments, the eukaryotic cells are yeast cells from a Saccharomycesspecies (e.g., Saccharomyces cerevisiae), a Hansenula species (e.g.,Hansenula polymorpha), a Yarrowia species (e.g., Yarrowia lipolytica), aKluyveromyces species (e.g., Kluyveromyces lactis) or a Pichia species(e.g., Pichia pastoris). According to a specific embodiment, theeukaryotic cells are Pichia cells, and in a most particular embodiment,Pichia pastoris cells. Pichia pastoris has been shown to have asecretory pathway with distinct Golgi stacks similar to those found inmammalian cells.

According to an alternative particular embodiment, the cells aremammalian cells selected from Hek293 cells or COS cells.

The eukaryotic (or specifically yeast) cells as described herein mayproduce uniformly, complex-type glycosylated glycoproteins that areready to use. Alternatively, the eukaryotic cells may produce twopopulations of easily separable, differentially glycosylatedglycoproteins, one population of which typically shows complex typeglycosylation, the other typically (though not necessarily) is singleGlcNAc-modified.

According to particular embodiments, the endoglucosaminidase enzymeencoded by the first exogenous nucleic acid sequence is amannosyl-glycoprotein endo-beta-N-acetylglucosaminidase, i.e., it hasthe activity of E.C. 3.2.1.96 in the IUBMB nomenclature, implying thatit can remove sugar chains while leaving one GlcNAc residue on theprotein. According to alternative embodiments, the endoglucosaminidaseencoded by the first exogenous nucleic acid sequence has differentaffinities toward different types of glycosylation structures. Typicalexamples of the latter are endoglucosaminidases that are able tohydrolyze hybrid type sugars and/or high-mannose sugars, but are notcapable of cleaving complex type glycans. According to furtherparticular embodiments, the endoglucosaminidase is amannosyl-glycoprotein endo-beta-N-acetylglucosaminidase that hasdifferent affinities toward different types of glycosylation structures.According to yet further particular embodiments, theendo-beta-N-acetylglucosaminidase is able to cleave hybrid type sugarsand/or high-mannose sugars, but not complex type glycans. According toeven more particular embodiments, the endoglucosaminidase is EndoH orEndoT. According to most particular embodiments, the endoglucosaminidaseis EndoT.

The glycoproteins produced by the cells described herein typicallyshould be easily recovered. This will particularly be achieved bysecretion of the glycoprotein. This can be after contact with theendoglucosaminidase (e.g., when the endoglucosaminidase remains in thecell), or before the contact with the endoglucosaminidase (e.g., whenboth are secreted). Secretion signals will in general be similar forboth glycoproteins and endoglucosaminidases (or optionally alsoglycosyltransferases), if the latter are secreted. The nature of thesecretion signal will indeed typically not depend on the protein to besecreted, but on the type of eukaryotic cells used. As long as thesecretion signal is functional in the cell type in which it is used(i.e., it results in secretion to the extracellular environment of theprotein or peptide to which it is fused), this feature is not criticalto the invention. Thus, secretion signals from other organisms may beused, as long as these signals lead to secretion in the eukaryotic cellsused. Secretion signals are well known in the art and may be derivedfrom—typically the N-terminus of—proteins that are secreted, or may bemade synthetically (e.g., Tan et al., Protein Engineering 2002, vol. 15,no. 4, pp. 337-345). Alternatively, they can be derived from genomicsequences using computational methods (Klee et al., BMC Bioinformatics2005, 6:256). Also, bacterial secretion signals can be used. Furtherexamples of signal peptides that can be used are described inWO2002/048187 (eukaryotic cells), Schaaf et al. (BMC Biotechnol. 2005;5:30) (moss cells), EP549062. Specific secretion signals used in yeastinclude, e.g., α-factor secretory peptide, the PH05 secretory peptide,and the BAR1 secretion signal.

Although secretion is particularly envisaged for easy recovery ofglycoproteins, alternative options exist. The produced glycoproteinsmay, for instance, be deposited in inclusion bodies in the cell, or inmembrane-bound organelles or in structures with similar functions. Whencells are part of an organism that is used for production (e.g., a plantinstead of a plant cell culture), the glycoprotein may be produced in ortransported to specific organs or tissues of the organism from which itcan be recovered (e.g., glands or trichomes). It should be noted that,particularly in cases where the protein is not secreted, it is possiblethat the protein is deposited in an inactive form. Thus, additionalrefolding or re-activating steps may be needed in order to obtain aphysiologically relevant form of the glycoprotein.

Although, in addition to the glycoprotein, the endoglucosaminidase mayalso be secreted by the cell (using identical or similar secretionsignals—i.e., the remarks on secretion signals for glycoproteins alsoapply for endoglucosaminidases), it can be a particular advantage thatthe endoglucosaminidase remains in the cell. This takes away the needfor separation of the endoglucosaminidase and the glycoprotein, whicharises when both proteins are secreted. Most particularly, theendoglucosaminidase not only remains in the cell, but is also fullyactive. Its activity should be regulated spatiotemporally in order toensure that the desired hydrolysis takes place. To this end, theendoglucosaminidase may be operably linked to an ER or Golgilocalization signal. Such signal directs the endoglucosaminidase to theER or Golgi, respectively, where it is retained. As the ER and Golgiapparatus are the intracellular locations where glycosylation ofproteins takes place, targeting to these organelles ensures that theendoglucosaminidase is in the correct intracellular position to modifythe glycosylation of the glycoprotein.

This is particularly also true for the glyco-engineered yeast cellsdescribed herein, as the at least one enzyme needed for complexglycosylation is also targeted to function in the ER→Golgi secretorypathway, the endoglucosaminidase can be targeted in such a way thatthese enzymes act cooperatively on the glycoprotein.

Indeed, in yeast—as in humans—the luminal surface of the ER and Golgiapparatus provides catalytic surfaces that allow the sequentialprocessing of glycoproteins as they proceed from the ER through theGolgi network into the medium. As a glycoprotein proceeds from the ERthrough the secretory pathway, it is sequentially exposed to differentmannosidases and glycosyltransferases. Several processing steps rely onprevious reactions because some N-glycosylation enzymes depend on aparticular substrate that is created by the previous enzyme.N-glycosylation enzymes, in particular, exogenous enzymes such as theendoglucosaminidase and the at least one enzyme needed for complexglycosylation, must therefore, be arranged in a predetermined sequenceto allow for the synthesis of specific N-glycan structures.

Establishing the sequential processing environments of the secretorypathway requires the proper localization of N-glycosylation enzymes. Themechanisms by which secreted proteins can be transported through thesecretory pathway (from the ER to the cis-, medial- and trans-Golgicompartments and into the medium), while each compartment maintains aspecific set of resident (for example, N-glycosylation) enzymes, hasbeen the subject of extensive study. Two well-established mechanismsthat localize proteins to the various compartments of the secretorypathway are retrieval and retention (van Vliet et al., PBMB 1 2003;Teasdale et al., 27 1996).

Retrieval is a process by which proteins are localized to certainorganelles through interaction with other proteins. Several ER-residingproteins contain a carboxy-terminal tetrapeptide with the consensussequence KDEL (SEQ ID NO:1) (or HDEL (SEQ ID NO:2) in yeast), which hasbeen shown to be required for efficient localization to the ER.

Several ER- and Golgi-residing enzymes are type II membrane proteins.These proteins have a common domain structure comprising a shortcytoplasmic tail at the amino terminus, a hydrophobic transmembranedomain, a luminal stem and a C-terminal catalytic domain. Deletionstudies as well as fusions to non-Golgi-residing proteins haveidentified the N-terminus and, in particular, the transmembrane region,as containing the targeting information of many type II membraneproteins. Although it is clear that N-terminal domains are involved intargeting, the extent to which their targeting ability is transferablebetween different species is not yet totally clear. Nevertheless,considerable advances have been made, such as the design of geneticlibraries of known type II membrane protein domains that encode peptidesthat are associated with proteins that naturally localize to the ER andGolgi of S. cerevisiae or P. pastoris (Choi et al., 5022 2003; Hamiltonet al., Science 1244) confirming the suitability of, e.g., the leadersequence from S. cerevisiae Sec12 (ER localization), MNN2 (Golgilocalization), and MNN9 (Golgi localization). Sequences listed in table5 of WO02/000879 include HDEL and the leader sequences from MnsI for ERlocalization, and leader sequences from Och1 and Mnt1 (Golgi-cislocalization), from Mnn2 (Golgi medial localization), from Mnn1 (Golgitrans localization), from alpha-2,6-sialyltransferase (trans-Golginetwork) and from beta-1,4-galactosyltransferase I (Golgi localization).

Localization signals thus are well known in the art and may be derivedfrom proteins that are normally localized in the ER or Golgi for theirfunction. Moreover, localization sequences from one organism mayfunction in other organisms. For example, the membrane-spanning regionof α-2,6-sialyltransferase from rats, an enzyme known to localize in therat trans Golgi, was shown to also localize a reporter gene (invertase)in the yeast Golgi (Schwientek, et al., 1995). Schwientek and co-workershave also shown that fusing 28 amino acids of a yeastmannosyltransferase (Mntl), a region containing an N-terminalcytoplasmic tail, a transmembrane region and eight amino acids of thestem region, to the catalytic domain of human GalT are sufficient forGolgi localization of an active GalT (Schwientek et al. 1995 J. Biol.Chem. 270 (10):5483-5489). Other well-documented motifs are the KDEL andHDEL motif for retention in the ER. According to particular embodiments,the ER or Golgi localization signal is from a protein that is itselflocalized in the ER or Golgi when functionally active. Examples of suchproteins include, but are not limited to, S. cerevisiae dipeptidylaminopeptidase A (Ste13p), human β-galactoside-α-2,6-sialyltransferase(ST6GalI) and the human ganglioside-GM₂-synthase. According to furtherembodiments, the localization sequence is derived from one of thefollowing proteins: Ste13p, GL2-synthase, ganglioside-GM₂-synthase, andα-2,6-glycosyltransferase, in particular α-2,6-sialyltransferase, mostparticularly, β-galactoside-α-2,6-sialyltransferase.

Importantly, the Golgi apparatus is not just one homogeneous region, buthas five functional regions: the cis-Golgi network, cis-Golgi,medial-Golgi, trans-Golgi, and trans-Golgi network. Vesicles from theendoplasmic reticulum (via the vesicular-tubular cluster) fuse with thecis-Golgi network and subsequently progress through the stack ofcisternae that make up the Golgi apparatus to the trans-Golgi network,where they are packaged and sent to the required destination. Eachregion contains different enzymes that selectively modify the contents,e.g., depending on where they are destined to reside. Thus, depending onthe exact targeting of the endoglucosaminidase within cells,glycosylation pathways may be modified in different ways.

For instance, the endoglucosaminidase may be targeted late in the Golgi,after sugar structures have already been added to the glycoprotein. Thismay, for instance, be particularly envisaged as a kind of “proofreading”or “in vivo clean-up,” i.e., in situations where the desired complexglycosylation pattern is produced on the glycoproteins as well as hybridtype and/or high mannose structures (a situation often observed inyeasts modified for human-type glycosylation). There, a late-Golgitargeting of an endoglucosaminidase specific for hybrid-type andhigh-mannose glycosylation structures (e.g., EndoT, EndoH) ensures thatthe aberrantly glycosylated glycoproteins are deglycosylated(particularly to a single GlcNAc), while the glycoproteins with complexglycosylation are secreted as such. Thus, two easily separableglycopopulations are obtained. An alternative option is the latetargeting of an endoglucosaminidase that hydrolyzes all glycosylationstructures made in the cell (which notably need not beendoglucosaminidases with broad specificity, as some eukaryotic cellshave only a limited glycodiversity, or as the eukaryotic cells may bemodified to produce glycoproteins with limited glycodiversity, e.g., bydeficiency of an enzymatic activity needed for complex glycosylation).This way, a uniform glycosylation pattern may be obtained in thepopulation of glycoproteins, e.g., only non-glycosylated or only singlemonosaccharide-modified glycoproteins. Another option would be to targetthe endoglucosaminidases to an earlier stage in the ER→Golgiglycosylation pathway, while a glycosyltransferase (e.g., an additionalexogenous glycosyltransferase that is targeted to later in the pathway)is active further downstream. This way, a uniform glycopopulation (e.g.,of single GlcNAc-modified glycoproteins) is presented as substrate tothe glycosyltransferases. This results in a uniform population ofglycosylated glycoproteins. Note that this uniform glycopopulation mayparticularly be a uniform population of non-naturally occurringglycoforms, as typical endoglucosaminidases will also remove the innerMan₃GlcNAc₂ core structure typical of natural glycostructures. However,such structures are often less immunogenic in mammals than particularglycans produced in plant, yeast or insect cells.

It will be clear that statements made here on the targeting ofendoglucosaminidases, of course, also apply to the targeting of otherenzymes within the cell, in particular, to glycosyltransferases and/orto the at least one enzyme needed for complex glycosylation used inparticular embodiments. Indeed, as these enzymes are active in theER→Golgi pathway and act sequentially, these enzymes should be carefullytargeted. According to particular embodiments, the at least one enzymeneeded for complex glycosylation is more than one enzyme. Moreparticularly, the at least one enzyme is the number of enzymes needed toform a pathway for complex glycosylation. Most particularly, each ofthese enzymes needed for complex glycosylation is targeted so that theyact sequentially and in the right order (typically, one enzyme willmodify the sugar chain to a substrate for the next enzyme). According toa particular embodiment, the at least one enzyme needed for complexglycosylation is at least one N-acetylglucosaminyl transferase (e.g.,GnT I, GnT II, GnT III, GnT IV, GnT V, GnT VI), at least one mannosidase(in particular mannosidase II), at least one fucosyltransferase, atleast one galactosyltransferase, at least one sialyltransferase, or anycombination of these enzymes.

Examples of glyco-engineered yeasts wherein complex glycosylationpathways have been engineered are extensively described in the art (see,e.g., Choi et al., 5022 2003; Hamilton et al.; Science 1244; Wildt etal., 119 2005; Hamilton et al., 387 2007; EP1211310; WO02/000879;US2006148039). Note that the enzyme(s) needed for complex glycosylationis/are all targeted to compartments of the secretory ER→Golgi pathwayand, thus, are not secreted.

In addition, a number of other genes may also be transformed in theglyco-engineered yeast cells described herein to ensure optimalproduction of complex-type glycosylated glycoproteins, such as ER andGolgi-specific transporters (e.g., sym- and antiport transporters forUDP-galactose and other precursors), or enzymes involved in thesynthesis of activated oligosaccharide precursors such as UDP-galactoseand CMP-N-acetylneuraminic acid. Indeed, the contacting with the atleast one enzyme needed for complex glycosylation may occur in thepresence of specific glycosyl donors (e.g., sugar nucleotide donors) toensure efficient and correct glycosylation.

The glycosylation status of the produced glycoprotein will depend bothon the cellular system used (e.g., which enzymes are present therein)and the specificity of the endoglucosaminidase. Moreover, the time andplace where these enzymes act is also important (e.g., which enzyme actsfirst in the ER→Golgi pathway). Thus, it is possible that cells willexpress solely non-glycosylated proteins, or proteins having only singleGlcNAc residues (e.g., in the case of yeast cells and anendoglucosaminidase capable of hydrolyzing high-mannose and hybrid typeglycans). These proteins can serve as the basis for, e.g.,crystallization studies. Another possibility is that such proteins arefurther modified, e.g., by treatment with glycosyltransferases,resulting in proteins with the desired glycan moieties.

Alternatively, cells can be used capable of achieving the desired(typically complex) glycosylation (e.g., glyco-engineered yeast whereinthe endoglucosaminidase acts after the enzymes needed for complexglycosylation (either intracellularly, e.g., in the trans Golgi ortrans-Golgi network, or extracellularly)). A prerequisite in thisscenario is that the endoglucosaminidase does not hydrolyze the desiredsugar chains (e.g., because of its specificity, because theendoglucosaminidase is spatially and/or temporally separated from theglycosylated protein, or because the endoglucosaminidase is renderedinactive after it has removed undesired glycans). Typically, such cellswill produce two populations of glycoproteins: the correctlyglycosylated form and a non-glycosylated or single GlcNAc-modified form(obtained, e.g., from deglycosylation of glycoproteins with hybrid-typeor mannose-type glycan modifications). Although such mixed populationstill requires a separation step before a uniformly glycosylatedpopulation is obtained, this separation step is much easier than withtraditional production methods, as the (e.g., weight, hydrodynamicproperties) difference between proteins with complex glycosylation andnon-glycosylated proteins is much larger than between differentlyglycosylated proteins.

Alternatively, it can be envisaged that the cells produce and/or secreteonly correctly glycosylated proteins. For, e.g., glyco-engineered yeast,this can be achieved by targeting the endoglucosaminidase enzyme justbefore the at least one enzyme for complex glycosylation in the ER→Golgipathway, in such a way that all glycoproteins are first (at leastpartly) deglycosylated by the endoglucosaminidase, after which they aremodified by the at least one enzyme for complex glycosylation. Using thelatter approach, the produced glycoproteins may have non-naturallyoccurring carbohydrate chains, as the endoglucosaminidase typically willremove the core Man₅GlcNAc₂ structure, or at least part thereof, so thatthe sugar chain added on the glycoprotein by the enzymes for complexglycosylation will be added on shortened base structures, such as asingle GlcNAc residue. Although not naturally occurring, such complexsugar chains often also are non-immunogenic and may have other desirableproperties, such as, e.g., increased stability, longer half-life, etc.Always important, but particularly in the generation of such new,synthetic pathways, is that the glycoprotein after modification by afirst enzyme (e.g., an endoglucosaminidase) is a suitable substrate forthe next enzyme (e.g., an enzyme needed for complex glycosylation).

However, it is understood that further (complex) glycosylation may alsobe inhibited, e.g., in order to retain solely non-glycosylated proteinsor single-monosaccharide-modified proteins. Thus, according to aparticular embodiment, the eukaryotic cells described herein do notcomprise at least one enzyme needed for complex glycosylation, such asER-mannosidase I, glucosidase I, glucosidase II, galactosyltransferase,sialyltransferase, mannosidase II, N-acetylglucosaminyl transferase I,and N-acetylglucosaminyl transferase II. Such cells are not capable ofcomplex glycosylation of glycoproteins. Nevertheless, even though(complete) complex glycosylation is normally not achieved in such cells,it may be possible to target an endoglucosaminidase with a particularspecificity to a place in the ER→Golgi glycosylation pathway where itensures that the glycoprotein after it has been contacted with theendoglucosaminidase is again a target for the following enzymes. Thisway, new synthetic pathways may be generated. It may, for instance, bepossible in a cell that lacks N-acetylglucosaminyl transferase I totarget an endoglucosaminidase just before the galactosyltransferase andsialyltransferase. This way, only the galactosyltransferase andsialyltransferase will act on the (partially) deglycosylated protein(e.g., a single-GlcNAc-modified protein), thus yielding a protein withnon-naturally occurring complex glycosylation.

Whereas cells for the production of glycoproteins as described hereinwill typically be provided in the form of a cell culture, this need notnecessarily be the case. Indeed, the cells producing the glycoproteinsmay be part of an organism, e.g., a transgenic animal or plant.According to a particular embodiment, plants comprising the glycoproteinand endoglucosaminidase-containing cells as described in the applicationare also envisaged. Typically, plants will have multiple of these cells,particularly also in different organs and/or tissues.

The eukaryotic cells described herein are particularly well suited forglycoprotein production. According to particular embodiments, theglycoproteins are enriched for a specific glycoform, particularly singleGlcNAc-modified glycoproteins. Thus, methods are provided for producingglycoproteins modified with a single GlcNac moiety in a eukaryotic cell,comprising the steps of:

-   -   providing a eukaryotic cell comprising a first exogenous nucleic        acid sequence encoding an endoglucosaminidase enzyme and a        second exogenous nucleic acid sequence encoding a glycoprotein        in conditions suitable for expressing the endoglucosaminidase        enzyme and the glycoprotein; and    -   recovering the glycoprotein after it has been intracellularly or        extracellularly contacted with the endoglucosaminidase.

Although the glycoproteins with a single GlcNAc residue may be the onlyglycoform of the glycoprotein produced by the cell (i.e., a uniformglycopopulation is produced), the methods may also be used to enrichsingle GlcNAc-modified proteins in a mixed population, or rather, toremove the glycoproteins with undesired glycosylation patterns byconverting them to single GlcNAc-modified proteins. Since singleGlcNAc-modified proteins are both easier to separate from a mixedglycopopulation as an easier starting point for furthertransglycosylation reaction, this is a considerable advantage. So eventhough several glycoforms of the glycoprotein may be produced, thesetypically can be easily separated (e.g., proteins with complexglycosylation as well as proteins with single GlcNAc residues).According to particular embodiments, the eukaryotic cells used in themethods described herein are not capable of complex glycosylation ofglycoproteins, or at least not of naturally occurring complexglycosylation of glycoproteins (i.e., with the inner Man₃GlcNAc₂ core).

The methods as described herein may be further adapted to ensure thatthe contact between glycoprotein and endoglucosaminidase occurs underoptimal circumstances (i.e., to ensure optimal activity of theendoglucosaminidase on the glycoprotein). For instance, when the contactoccurs intracellularly, the endoglucosaminidase may be targeted to the(desired place in the) Golgi or ER where it exerts its function on theglycoprotein. Depending on, e.g., further transglycosylation envisagedin or outside the cell, the desired place may vary, as described above.According to particular embodiments, the intracellular contact occurs inthe Golgi or ER.

Both the endoglucosaminidase and the glycoprotein may also be secretedand the contact may happen extracellularly. Depending on the cells andendoglucosaminidase that are used, however, the optimal growth andproduction conditions for the cells (e.g., pH, temperature) may differfrom the optimal conditions for enzymatic activity. Thus, the mediumwhere the extracellular contact between the glycoprotein and theendoglucosaminidase takes place may be adjusted for optimal enzymaticactivity of the endoglucosaminidase. According to a particularembodiment, the conditions of the medium wherein the extracellularcontact takes place are adjusted for optimal enzymaticendoglucosaminidase activity. According to a further particularembodiment, the pH of the medium wherein the extracellular contact takesplace is adjusted for optimal enzymatic endoglucosaminidase activity.Typically, this may be done by a pH shift of the medium after the cellshave been allowed to produce and secrete both glycoproteins andendoglucosaminidases. In general, such pH shift will be a downshift, asendoglucosaminidases usually are physiologically active in an acidicenvironment. According to another particular embodiment, the temperatureof the medium is adjusted for optimal enzymatic activity. Note that theadjustment of growth and production conditions may be done just beforeendoglucosaminidase activity, or that the conditions may already beenadapted during cell growth. For instance, Pichia cells can grow andproduce proteins in a fairly acidic medium, which thus is alreadyadjusted for optimal activity of particular endoglucosaminidases.However, as some eukaryotic cells are dependent on N-glycosylation fortheir integrity, it might be beneficial to buffer the pH of the growthmedium to a pH at which the endoglucosaminidase is not active, anddown-shift the pH only after the protein production is finished.

According to a particular aspect, the protein modified with the singleGlcNAc residue is only an intermediary product. Methods according tothis aspect will include at least one additional transglycosylationstep, which can occur both extracellularly (via an added enzyme, or viaan enzyme also produced by the cells) or intracellularly. According tothese embodiments, before the final recovery of the glycoprotein, themethods further involve a step of contacting the enzyme with aglycosyltransferase after it has been intracellularly or extracellularlycontacted with the endoglucosaminidase. Optionally, this contacting witha glycosyltransferase may occur in the presence of (potentially extraadded) specific glycosyl donors (e.g., sugar nucleotide donors) toensure efficient and correct glycosylation. This will especially be thecase when the transglycosylation takes place extracellularly.

If the transglycosylation step takes place intracellularly, it will beunderstood by the skilled person that, when both the endoglucosaminidaseenzyme and the glycosyltransferase enzyme are targeted to the ER orGolgi, it is ensured that the glycosyltransferase activity occurs afterthe endoglucosaminidase activity. Typically, this may be ensured bytargeting both enzymes to different compartments of the ER or Golgi, asthere is a fixed order in which proteins follow the ER→Golgi route. Inthe event both enzymes are targeted to the same compartment, or thatboth activities are performed by the same enzyme, it typically will beensured that the protein after the transglycosylation step is no longerrecognized as substrate for the endoglucosaminidase enzyme. Thus,separation of the enzymatic activities in time may also involve spatialseparation and/or a different substrate specificity and/or inactivationof the enzyme. According to a particular embodiment, both theendoglucosaminidase and the glycosyltransferase are produced by the samecell, but only the glycosyltransferase is secreted, to ensuretransglycosylation takes place after the endoglucosaminidase activity.

Depending on how the method is performed, the glycosyltransferase enzymemay be added extracellularly (i.e., is not produced by the same cells),is also produced and secreted by the cells producing the glycoproteinand endoglucosaminidase, or is also produced by the cells and retainedwithin the ER or Golgi apparatus. The glycosyltransferase may be encodedby an exogenous sequence, or may be an enzyme that is endogenous in thecells having a first exogenous nucleic acid sequence encoding anendoglucosaminidase enzyme and a second exogenous nucleic acid sequenceencoding a glycoprotein.

According to particular embodiments using glyco-engineered yeast asdescribed herein, the glycoproteins are enriched for a specific(complex-type) glycoform, while proteins with high-mannose type andhybrid-type glycosylation are depleted by hydrolyzing the glycans tosimpler forms (e.g., a single GlcNAc residue). Thus, methods areprovided for producing glycoproteins in a glyco-engineered yeast cellwhile depleting proteins with high mannose-type glycosylation and/orhybrid-type glycosylation, comprising the steps of:

-   -   providing a glyco-engineered yeast cell comprising a first        exogenous nucleic acid sequence encoding an endoglucosaminidase        enzyme, a second exogenous nucleic acid sequence encoding a        glycoprotein, and at least a third exogenous nucleic acid        sequence encoding at least one enzyme needed for complex        glycosylation, selected from the group consisting of        N-acetylglucosaminyl transferase I, N-acetylglucosaminyl        transferase II, mannosidase II, galactosyltransferase, and        sialyltransferase, in conditions allowing expression of the at        least three nucleic acid sequences; and    -   recovering the glycoprotein after it has been intracellularly        contacted with the at least one enzyme needed for complex        glycosylation and intracellularly or extracellularly contacted        with the endoglucosaminidase.

“Contacted” as used herein does not only refer to physical proximity,but specifically implies that the enzyme with which the glycoprotein iscontacted has the opportunity to exert its enzymatic function on theglycoprotein. Thus, physical proximity to an inactive, temporallyinactive or inactivated enzyme does not constitute “contact” as definedherein—this requires contact with an active enzyme in both aconformation (i.e., spatial orientation and distance between theproteins) and time-frame that are sufficient for enzymatic activity.

Depleting proteins with high mannose-type glycosylation and/orhybrid-type glycosylation in yeast cells (by selectively convertingthese glycoforms to, e.g., single GlcNAc-modified proteins) may resultin yeast cells producing glycoproteins as a uniform and homogeneous,typically complex, glycopopulation. Alternatively, several glycoforms ofthe glycoprotein may be produced, but these typically can be easilyseparated as no glycoproteins with sugar chains of comparable size tothe complex glycans are produced. An example of mixed glycoforms thatare produced are proteins with complex glycosylation as well as proteinswith single GlcNAc residues. The single GlcNAc-modified proteinsthemselves can be used, e.g., as starting point for furthertransglycosylation reactions, to result in proteins with complexglycosylation, or can be used as such in crystallization studies.

The methods as described herein may be further adapted to ensure thatthe contact between glycoprotein and endoglucosaminidase occurs underoptimal circumstances (i.e., to ensure optimal activity of theendoglucosaminidase on the glycoprotein). For instance, when the contactoccurs intracellularly, the endoglucosaminidase may be targeted to the(desired place in the) Golgi or ER where it exerts its function on theglycoprotein. The same, of course, applies for the contact between theglycoprotein and the at least one enzyme for complex glycosylation.Depending on the specific order envisaged (in particular, whether theendoglucosaminidase is contacted with the glycoprotein before or afterthe contact with the enzyme(s) needed for complex glycosylation), thedesired place within the ER or Golgi (e.g., cis-Golgi network,cis-Golgi, medial-Golgi, trans-Golgi, and trans-Golgi network) may vary,as described above. According to particular embodiments, theintracellular contact with the at least one enzyme needed for complexglycosylation occurs in the Golgi or ER. According to particularembodiments, the intracellular contact with the endoglucosaminidaseoccurs in the Golgi or ER. According to further particular embodiments,the glycoprotein is contacted with the endoglucosaminidase before it iscontacted with the at least one enzyme needed for complex glycosylationin the ER→Golgi secretory pathway. According to alternative furtherparticular embodiments, the contact between glycoprotein andendoglucosaminidase occurs in the ER or Golgi, but after the contactwith the at least one enzyme needed for complex glycosylation. Accordingto yet further particular embodiments, the respective targeting signalsof the endoglucosaminidase and the enzyme needed for complexglycosylation are chosen in such a way that the enzymes are targeted todifferent functional regions (ER, cis-Golgi network, cis-Golgi,medial-Golgi, trans-Golgi, and trans-Golgi network) so that they actsequentially. According to still further particular embodiments, theenzymes are targeted in such a way that they act immediately after eachother, e.g., they may be targeted to adjacent compartments in the Golgiapparatus.

Unlike the at least one enzyme needed for complex glycosylation, theendoglucosaminidase may also be secreted. This may be the case when theglycoprotein is also secreted and the contact between glycoprotein andendoglucosaminidase happens extracellularly (after the intracellularcontact with the at least one enzyme needed for complex glycosylation).Depending on the cells and endoglucosaminidase that are used, however,the optimal growth, production and secretion conditions for the cells(e.g., pH, temperature) may differ from the optimal conditions forenzymatic activity. Typically, the culturing of yeast cells happens atmore or less neutral pH (i.e., around pH 7), while the pH optimum ofseveral glycosidases is acidic (typical examples include enzymes withoptimum around pH 5 or a pH optimum of about 6). Thus, the medium wherethe extracellular contact between the glycoprotein and theendoglucosaminidase takes place may be adjusted for optimal enzymaticactivity of the endoglucosaminidase. According to a particularembodiment, the conditions of the medium wherein the extracellularcontact takes place are adjusted for optimal enzymaticendoglucosaminidase activity. According to a further particularembodiment, the pH of the medium wherein the extracellular contact takesplace is adjusted for optimal enzymatic endoglucosaminidase activity.Typically, this may be done by a pH shift of the medium after the cellshave been allowed to produce and secrete both glycoproteins andendoglucosaminidases. In general, such pH shift will be a downshift, asendoglucosaminidases usually are physiologically active in an acidicenvironment. According to particular embodiments, the culturing of theyeast cells and production and secretion of the glycoprotein occur at amore or less neutral pH, in particular between pH 6 and 8, more inparticular between pH 6.5 and pH 7.5, even more in particular between pH6.7 and 7.3, most in particular at pH 7. According to specificembodiments, the extracellular contact between glycoprotein andendoglucosaminidase occurs at a pH of between 4 and 6, more inparticular between pH 4.5 and pH 5.5, even more in particular between pH4.7 and pH 5.3, most in particular at pH 5. According to an alternativeembodiment, the contacting occurs between pH 4 and 5, pH 4.5 and 5 orbetween pH 4.7 and 5.

According to a specific combination of embodiments, the pH of the mediumis adjusted after the growth and production/secretion phase to provideoptimal conditions for the endoglucosaminidase. According to particularembodiments, the pH is downshifted. According to further particularembodiments, the pH shift is at least 0.5 units, at least 1 unit, atleast 1.5 units or at least 2 units. According to specific embodiments,the pH is shifted from between pH 6 and 8 for growing conditions betweenpH 4 and 6 for the contacting with the enzyme and enzymatic activity.According to alternative embodiments, however, cells are grown inconditions that are permissive for both growth/production and enzymaticactivity. For instance, the yeast Pichia pastoris is able to grow andproduce proteins at lower pH (e.g., pH 5), which is the pH optimum forenzymes such as endoH or, in particular, endoT. Similarly, if yeastcells are chosen that have limiting conditions for optimal growth, it ispossible to choose an endoglucosaminidase enzyme with a broad optimumrange.

According to another particular embodiment, the temperature of themedium is adjusted for optimal enzymatic activity. Note that theadjustment of growth and production conditions may be done just beforeendoglucosaminidase activity, or that the conditions may already beenadapted during cell growth. As already mentioned, Pichia cells can growand produce proteins in a fairly acidic medium, which, thus, is alreadyadjusted for optimal activity of particular endoglucosaminidases.

It is to be understood that although particular embodiments, specificconfigurations as well as materials and/or molecules, have beendiscussed herein for cells and methods according to the disclosure,various changes or modifications in form and detail may be made withoutdeparting from the scope and spirit of the disclosure. The followingexamples are provided to better illustrate particular embodiments, andthey should not be considered limiting the application. The applicationis limited only by the claims.

EXAMPLES Example 1 Intracellular and Soluble Expression of a Trichodermareesei endo-N-acetyl-β-D-glucosaminidase (EndoT)) in Pichia pastorisIntroduction and Strategy

Saprophytic filamentous fungi produce and secrete a variety ofhydrolases needed for the degradation of organic material. Inparticular, organisms secreting cellulases and hemicellulases are ofgreat interest to the biotechnological industry and can be used indegradation of biomass for, e.g., bio-fuel production. One of the bestproducers of such enzymes is Trichoderma reesei.

It was shown previously that the glycosylation pattern on T. reeseisecreted proteins varies considerably depending on the environmentalconditions. Many of the differences in glycosylation are attributable topost-secretory trimming events by extracellular hydrolases, eitherbecoming post-translationally activated or being differentiallyregulated on transcription level because of the applied growthconditions. Very peculiar in this sense, is the presence of only asingle GlcNAc-residue onto the Asn of known N-glycosylation sites.However, recent findings clearly indicate that this is the result of anendo-N-acetylglucosaminidase-like activity, here called EndoT, which hasnow been successfully purified from the T. reesei growth medium (see,WO2006/050584).

Enzymes acting on the chitobiose part of N-linked glycans, likeendo-N-acetyl-β-D-glucosaminidases (e.g., EndoH) andN-linked-glycopeptide-(N-acetyl-beta-D-glucosaminyl)-L-asparagineamidohydrolases (e.g., PNGase F) are important tools in the isolationand analysis of oligosaccharides from glycoproteins. Moreover,glycosidases that are able to deploy deglycosylation activities on anative protein (such as EndoH) have proven to be invaluable for theelucidation of the crystal structure from several glycoproteins.Purified T. reesei EndoT was proven to be able to act upon high-mannoseand hybrid, but not on complex N-glycans from native proteins.

Based on internal peptide sequence information, the gene encoding EndoTcould be deduced in silico. However, when comparing results from N- andC-terminal sequence analysis, SDS-PAGE and iso-electric focusing on theone hand and in silico ORF translation, and following MW/pI calculationson the other hand, it was clear that—apart from the cleavage of apredicted 17 amino acid signal peptide—further proteolysis occurs atboth the N- and C-terminus of the protein. At the time it was not knownwhether this happens intracellularly and/or extracellularly, and whetherthese proteolytic steps are important for protein maturation and maximalenzyme activity.

Therefore, soluble expression of several forms of processed EndoT, i.e.,the mature protein (EndoT[FullSize]), the mature protein missing nineextra N-terminal amino acids (EndoT[-Nterm]), the mature protein missing43 C-terminal amino acids (EndoT[-Cterm]) and the mature protein missingboth the N- and C-terminal amino acids (EndoT[-N/Cterm])—was establishedin the methylotrophic yeast Pichia pastoris. The four forms werepurified from the medium and their specific activity was determined.Moreover, the EndoT activity was also locally expressed in the latecompartments of the Pichia secretion pathway by fusing EndoT[FullSize]to the localization signal of S. cerevisiae dipeptidyl aminopeptidase A(Ste13p), a protein known to reside within the yeast trans-Golgi network(Nothwehr et al., 1993). With this, we envisage the clean-up ofnon-complex N-glycans produced within a glyco-engineered expressionstrain of Pichia pastoris, before secretion of the recombinantglycoproteins into the cultivation broth.

Materials and Methods:

Strains and Growth Conditions

Plasmid construction and propagation was performed using chemocompetentEscherichia coli MC1061 cells (hsdR2 hsdM⁺ hsdS⁺ araD139 Δ(araleu)₇₆₉₇Δlac_(X74) galE15 galK16 rpsL (St^(r)r) mcrA mcrB1) (Casadabanand Cohen, 1980). Growth and transformation of E. coli was done viastandard procedures (Sambrook et al., 1989).

The following Pichia strains were used during the experimental set-up:GS115 (his4) (Invitrogen), GS115-Man5 (his4) and GS115-hIFNβ (HIS4).GS115-Man5 is a derivative of GS115, transformed with pGlycoSwitch-M5and mainly synthesizing Man₅GlcNAc₂ N-glycans on its secreted proteins(Vervecken et al., 2004; Vervecken et al., 2007). GS115-hIFNβ (HIS4) isa derivative of GS115, transformed with pPIC9hIFNβ and secreting humaninterferon beta (hIFNβ). For protein production purposes, yeast strainswere pregrown in BMGY medium (1% yeast extract, 2% peptone, 1% glycerol,1.34% yeast nitrogen base w/o amino acids and 100 mM potassium phosphatepH 6.0) for 48 hours at 30° C. and 250 rpm while protein expression wasinduced after transfer of the cells into BMMY (1% yeast extract, 2%peptone, 1% methanol, 1.34% yeast nitrogen base w/o amino acids and 100mM potassium phosphate pH 6.0) and further cultivation at 30° C. and 250rpm.

The S. cerevisiae strain INVSc1 (α, leu2-3, 112 his3Δ1, trp1-289,ura3-52) (Invitrogen) was used to prepare genomic DNA as a template forthe amplification of specific STE13 gene fragments (see, below). Generalmaintenance of strain INVSc1 as well as the different Pichia strains andtransformants was done on YPD (1% yeast extract, 2% pepton, 2%dextrose).

Plasmid Construction

A custom-made, codon-optimized synthetic gene was ordered at GeneArt AG(Regensburg, Germany) for the expression of mature EndoT in Pichiapastoris. At the 5′ site, an EcoRI restriction site followed by thesequence CTC GAG AAA AGA GAG GCT GAA GCG (SEQ ID NO:3)—encoding theC-terminal part of the S. cerevisiae alpha-mating factor pro-region andthe Kex2p cleavage site (Leu-Val-Lys-Arg-Glu-Ala-Glu-Ala) (SEQ IDNO:4)—were introduced for easy downstream cloning purposes. A fewexceptions to the optimal Pichia codon usage were requested for theintroduction of specific unique restriction sites: Ala8-Val9-Pro10(counting starts from the first codon of the alpha-mating factorpro-region part (CTC encoding Leu)) is encoded by GCG GTA CCC for theintroduction of a KpnI site (underlined); Leu14-Gln15 is encoded by CTGCAG for the introduction of a PstI site; Pro24-Arg25 is encoded by CCTAGG for the introduction of an AvrII site; Glu307-Leu308 is encoded byGAG CTC for the introduction of an Ecl136II site and Arg339-Pro340 isencoded by AGG CCT for the introduction of a StuI site. The last codonof the mature EndoT (GCT encoding Ala350) is followed by the sequenceTAA CCC TAA GGT AAG CTT (SEQ ID NO:5), containing two stop codons (initalics) and the unique restriction sites Bsu36I respectively HindIII(underlined). The synthetic gene was provided as an AscI/PacI fragmentwithin the pGA18 vector backbone. From there, it was transferred as anEcoRI/HindIII fragment into pUC19, digested with the same enzymes, toresult into pUC19EndoT[FullSize]. Vector pUC19EndoT[-Nterm] wasgenerated by treating pUC19EndoT[FullSize] sequentially with KpnI, T4polymerase and AvrII to allow the integration of a blunt/AvrII-stickyadaptor sequence consisting of the sense oligonucleotide5′-GCCGAGCCGACGGACCTGC-3′ (SEQ ID NO:6) and the antisenseoligonucleotide 5′-CTAGGCAGGTCCGTCGGCTCGGC-3′ (SEQ ID NO:7). VectorpUC19EndoT[-Cterm] was constructed by treating pUC19EndoT[FullSize]sequentially with Bsu36I, Klenow polymerase and Ecl136II, and closing ofthe corresponding vector fragment using T4 DNA ligase.

To obtain Pichia plasmids for the soluble expression of the differentEndoT variants, the three pUC19-derived vectors were used to isolateEndoT[FullSize], EndoT[-Nterm] and EndoT[-Cterm] as a XhoI/NotIfragment. These fragments were introduced into a XhoI/NotI digestedpPIC9 vector, resulting in the Pichia expression plasmidspPIC9EndoT[FullSize], pPIC9EndoT[-Nterm] and pPIC9EndoT[-Cterm]respectively, in which the EndoT variants are cloned in-frame with thecomplete prepro-region of the S. cerevisiae alpha-mating factor. Togenerate vector pPIC9EndoT[-N/Cterm], an AvrII/NotI fragment of plasmidpPIC9EndoT[-Cterm] was isolated and cloned into an AvrII/NotI openedvector fragment of pPIC9EndoT[-Nterm]. Finally, pUC19EndoT[FullSize] wasused as a template to construct an expression plasmid for EndoT,containing an internal Kex2 cleavage site. First, pUC19EndoT[FullSize]was digested with Ecl136II and a phosphorylated double-stranded linkersequence encoding for Lys-Arg-Glu-Ala-Glu-Ala (SEQ ID NO:8)(5′-AAGAGAGAGGCTGAGGCC-3′ (SEQ ID NO:9)) was introduced. Then, theresulting EndoT[FullSize+Kex2] sequence was isolated from the pUC19backbone as a XhoI/NotI fragment and ligated into a XhoI/NotI openedpPIC9 template to generate pPIC9EndoT[FullSize+Kex2].

Plasmids for the intracellular expression of EndoT[FullSize] weregenerated by exchanging the prepro-region of the alpha mating factor forthe coding sequence of the first 140 or 240 N-terminal amino acids of S.cerevisiae Ste13p (dipeptidyl aminopeptidase A). These sequences werePCR-amplified using genomic DNA (gDNA) from strain INVSc1 as a template.The gDNA was prepared from an overnight yeast culture, grown in YPD at30° C. and 250 rpm, using the Nucleon Kit for extraction of yeast gDNA(GE Healthcare). Sense primer 5′-GGAATTCATGTCTGCTTCAACTCATTCG-3′ (SEQ IDNO:10) (underlined: EcoRI site) and antisense primer5′-CGGGGTACCGGTATTAGAATAACAAGTAGAAC-3′ (SEQ ID NO:11) (underlined: KpnIsite; in italics: codon for Pro140 of Ste13p) were used to amplify thegene fragment encoding the first 140 N-terminal Ste13p amino acids(i.e., the cytoplasmic and transmembrane regions of Ste13p), while thesame sense primer and antisense primer5′-CGGGGTACCGTAAATTCTACTCCTTCATATAGG-3′ (SEQ ID NO:12) (underlined: KpnIsite; in italics: codon for Thr240 of Ste13p) were used to generate agene fragment encoding the first 240 N-terminal Ste13p amino acids (thuscontaining 100 extra amino acids of the luminal domain of Ste13p). ThePCR reactions were performed using TaKaRa Ex Taq™ polymerase (TaKaRa BioInc.) at an annealing temperature of 56° C. The generated fragment weredigested with KpnI and EcoRI and cloned into the EcoRI/KpnI openedplasmid pUC19EndoT[FullSize], resulting inpUC19Ste13(140Aa)EndoT[FullSize] and pUC19Ste13(240Aa)EndoT[FullSize].After sequencing to check for PCR errors, these vectors were cut withEcoRI and NotI to isolate the Ste13p-EndoT fusion constructs. Theobtained fragments were cloned into an EcoRI/NotI digested pPICZAbackbone, resulting in the plasmids pPICZSte13(140Aa)EndoT[FullSize] andpPICZSte13(240Aa)EndoT[FullSize] respectively.

Pichia Transformation

Plasmids pPIC9EndoT[Full Size], pPIC9EndoT[-Nterm], pPIC9EndoT[-Cterm]and pPIC9EndoT[-N/Cterm] were linearized in the HIS4 selection markerusing SalI and transformed to P. pastoris GS115 (his4) (Invitrogen) andGS115-Man5 (his4) via electroporation (Cregg and Russell, 1998).Transformants were selected on minimal medium without histidine (2%dextrose, 0.67% yeast nitrogen base w/o amino acids, 1 M sorbitol, 0.77g/1 CSM-His (Bio101)).

Plasmids pPICZSte13(140Aa)EndoT[FullSize] and pPICZSte13(240Aa)EndoT[FullSize] were linearized in the 5′AOX1 promoter region and transformedto P. pastoris GS115-hIFNβ (HIS4). Transformants were selected on YPDcontaining 100 μg/ml of zeocin (Invitrogen).

Protein Analysis

Expression of secreted proteins was checked via standard SDS-PAGEanalysis and Coomassie staining Strains were pregrown in BMGY andprotein production was induced after transfer of the cells into BMMY, asindicated in the results section. Proteins were precipitated from themedium via the standard DOC/TCA procedure and the resulting proteinpellet was resuspended in 2× Laemmli buffer. The protein samples wereincubated for 5 minutes at 100° C. before loading on gel.

PNGaseF treatment of glycoproteins and the analysis of thedeglycosylated proteins were done as follows. Proteins from theinduction medium were precipitated with 2 volumes of ice-cold aceton.After incubation on ice for 20 minutes and centrifugation (14,000 rpm, 5minutes), the supernatant was removed and the protein pellet wasresuspended in 100 μl 50 mM Tris.HCl pH 8. SDS and β-mercaptoethanolwere added to a final concentration of 0.5 and 1% respectively. Sampleswere incubated for 5 minutes at 100° C., after which G7 buffer (10×buffer, New England Biolabs), NP-40 (final concentration of 1%),complete protease inhibitor (Roche) and in-house produced PNGaseF (1000units) were added. After overnight incubation at 37° C., proteins wereprecipitated via the DOC/TCA procedure, resuspended in 2× Laemmli bufferand analyzed via SDS-PAGE.

Enzymatic activity of EndoT was checked via a gel-shift analysis usingSDS-PAGE. Pichia medium containing one of the EndoT forms is incubatedat 30° C. in 50 mM NaOAc pH 5 in the presence of a glycoprotein. Afterdifferent time points, the proteins in the reaction mixture wereprecipitated by addition of 3 volumes of ice-cold 100% ethanol and a1-hour incubation on ice. After centrifugation (5 minutes, 14,000 rpm),the protein pellet was resuspended in 2× Laemmli buffer. The proteinsamples were incubated for 5 minutes at 100° C. before loading on gel.Higher mobility of the test glycoprotein serves as an indication for thedeglycosylation capacity of the produced EndoT. The enzymatic activityof the soluble EndoT forms was checked using either RNaseB (Sigma),fetuin from fetal calf serum (Sigma) or in-house produced T. reeseiα-1,2-mannosidase as test glycoproteins. Intracellular activity waschecked via co-expression of EndoT in a Pichia strain that secreteshuman IFNβ or T. reesei α-1,2-mannosidase.

N-Glycan Analysis

N-linked oligosaccharides were analyzed via DNA sequencer-assisted (DSA)fluorophore-assisted carbohydrate electrophoresis (FACE) using an ABI3130 capillary DNA sequencer (Laroy et al., 2006). N-glycans wereobtained by incubation of the EndoT forms with a glycoprotein, asdescribed for the enzymatic assay (see, above). After the reaction, theproteins are precipitated with 3 volumes of ice-cold 100% ethanol. Thesupernatant containing the N-glycans is separated from the proteinpellet and evaporated. The thus-obtained dried oligosaccharides arefurther treated (labeling with APTS and clean-up) and analyzed asdescribed (Laroy et al., 2006).

Results on Soluble Expression:

A Pichia expression construct was made for the soluble expression of thefour EndoT forms. For this, the coding sequences of the EndoT forms,fused in-frame to the prepro region of the S. cerevisiae alpha matingfactor, were placed under the transcriptional control of themethanol-inducible AOX1 promoter. The resulting plasmids weretransformed to P. pastoris GS115 (his4) and a glyco-engineered strain(Man5 strain) (his4) mainly synthesizing Man₅GlcNAc₂ N-glycans on itssecreted glycoproteins, and transformants were selected via theirability to grow on minimal medium without histidine. Expression levelsof EndoT were checked after falcon cultivation of several transformants:selected single clones were grown for 48 hours on BMGY (bufferedglycerol medium) to high cell density, after which protein expressionwas induced for 40 hours upon a transfer to BMMY (buffered mediumcontaining methanol as sole carbon source). Proteins were DOC/TCAprecipitated from 0.5 to 1 ml of the harvested growth medium,resuspended in 2× Laemmli loading buffer and analyzed via SDS-PAGE. Verystrong expression was observed for EndoT[FullSize] and EndoT[-Nterm],whereas the secreted levels of the forms lacking the 43 C-terminal aminoacids were significantly lower (data not shown). Expected MWs for theprotein backbone are 37.4 kDa for EndoT[FullSize], 36.4 kDa forEndoT[-Nterm], 32.7 kDa for EndoT[-Cterm] and 31.7 kDa forEndoT[-N/Cterm].

The lower expression levels of the C-terminally truncated EndoT formsmight be due to inefficient folding in the ER. In an attempt to improvethe secretion of these EndoT forms, an expression construct wasgenerated where a Kex2 cleavage recognition site(Lys-Arg-Glu-Ala-Glu-Ala (SEQ ID NO: 13)) was introduced after the codonfor the last amino acid of the C-terminally truncated EndoT. In thisway, a full-size version of the EndoT (now containing an internal Kex2site) gets translated and folded within the ER lumen. We assume, basedon the large expression of EndoT[FullSize], that this would be anefficient process. Once completely folded, truncation of EndoT can occurvia Kex2 cleavage in the late Golgi compartment of the Pichia cells.Since truncation of EndoT by Trichoderma reesei proteases is a naturalprocess, we assume that the introduced protease cleavage site will alsobe accessible for Pichia Kex2p. After introduction of the expressionplasmid into P. pastoris GS115 (his4) and the glyco-engineered strain(Man5 strain) (his4), transformants were selected via their ability togrow on minimal medium without histidine. Expression levels of truncatedEndoT were checked after falcon cultivation of several transformants asdescribed above. Proteins were DOC/TCA precipitated from 1 ml of theharvested growth medium and analyzed via SDS-PAGE. However, thisstrategy did not result in a significant increase of production ofC-terminally truncated EndoT by Pichia (data not shown). This couldindicate that the introduction of six extra amino acids might as wellresult into folding problems for the full-size version of the EndoT.

The enzymatic activity was initially checked for EndoT[FullSize] via anSDS-PAGE gel-shift analysis: a glycoprotein was incubated at 30° C. withPichia Man5 medium containing soluble EndoT[FullSize] in a NaOAc pH 5.0buffer and the degree of deglycosylation was checked on gel. Theglycoproteins under investigation were Pichia secreted in-house producedT. reesei α-1,2-mannosidase carrying high mannose core and hyperglycosylstructures, fetuin carrying complex N-glycans and RNaseB carrying highmannose (Man₅₋₉GlcNAc₂) structures. Incubations were performed withincreasing amounts of EndoT (1, 5 and 10 μl medium) and increasingamounts of time (1 hour, 3 hours and 20 hours).

Deglycosylation could be observed via SDS-PAGE analysis in the case ofα-1,2-mannosidase and RNaseB, but not fetuin. Trimming of themannosidase basically depends on the amount of EndoT added: treatmentwith 10 μl medium results in efficient deglycosylation after even 1 hourof incubation, whereas prolonged incubation with only 1 μl of mediumdoes not increase the efficiency of N-glycan trimming. In contrast,deglycosylation of RNaseB happens more in a time-dependent rather than aconcentration-dependent way. All together, the results indicate that thePichia produced EndoT[FullSize] is secreted as an active protein, actingon high-mannose but not on complex N-glycan structures. Moreover, themode of action on high-mannose N-glycans can differ, either depending onthe protein substrate (RNaseB versus α-1,2-mannosidase) or the type ofhigh-mannose N-glycans (core type versus hypermannosylation).

When expressed by Pichia, EndoT can deglycosylate itself. This wasclearly observed when analyzing GS115 and Man5 produced EndoT[FullSize]on the same gel, with and without preceding in vitro PNGaseF treatment(data not shown). Whereas glycosylated endoT was observed when secretedfrom wild-type Pichia pastoris, different glycoforms were observed inthe Man5 strain, which is the result of a partial deglycosylation event,importantly demonstrating that endoT can deglycosylate proteinsco-secreted in the growth medium (in this case, other endoT proteinmolecules).

The activity of the other forms of EndoT (expressed by the Man5 strain)was also monitored via a gel-shift analysis on α-1,2-mannosidase.Samples were incubated overnight with Pichia medium in NaOAc pH 5. Thisshowed that the truncated forms also have the potential to deglycosylatea given glycoprotein, albeit that the EndoT[-N/Cterm] protein issomewhat less effective (data not shown).

The analyses above strongly indicates that the N- and C-terminal aminoacids are not necessary for (at the very least basal) deglycosylationactivity. The low expression levels of the EndoT forms lacking theC-terminal amino acids, suggest that these amino acids might beimportant though for efficient protein folding upon translocation intothe ER.

The N-glycans, liberated from RNaseB after treatment with differentpurified forms of EndoT, were APTS-labeled and analyzed via capillaryelectrophoresis. The results were compared with those obtained afterRNaseB deglycosylation using EndoH and PNGaseF. The data show thatMan₅₋₉GlcNAc N-glycans were released from RNaseB using EndoH and thedifferent EndoT forms, while Man₅₋₉GlcNAc₂ structures were obtainedusing PNGaseF (FIG. 1). Thus, the specificity of EndoT resembles that ofEndoH.

Results on Intracellular Expression:

Two constructs, based on the localization signal of yeast Ste13p, weregenerated for the expression of Golgi-resident EndoT[FullSize]. In afirst construct, the 140 N-terminal amino acids of Ste13p, comprisingthe transmembrane region and the cytosolic domain known to containsignals for Golgi-localization, were fused to the first amino acid ofEndo[FullSize] (=fusion construct 1). A second construct was generatedas well where the first 240 N-terminal amino acids from Ste13p, so alsocomprising 100 amino acids of the Ste13p luminal domain, were fused toEndoT[FullSize] (=fusion construct 2).

The coding sequences of the fusion proteins were put under thetranscriptional control of the methanol-inducible Pichia AOX1 promoterand the resulting plasmids were transformed to Pichia GS115, expressinghuman interferon-beta (hIFNβ) or T. reesei α-1,2-mannosidase.Transformants were selected by their ability to grow on zeocin. HumanIFNβ contains one N-glycosylation site and is produced by Pichiapastoris as a mixture of a glycosylated and a non-glycosylated form,which are easily distinguishable from one another on a 15%poly-acrylamide gel. For each construct, eight single clones were grownfor two days in 100 ml shake flasks containing 30 ml BMGY. Once highcell densities were reached, the expression of soluble hIFNβ andintracellular EndoT was induced upon transfer to BMMY medium. Proteinsfrom 0.5 ml medium, taken after 24 and 40 hours of induction, wereDOC/TCA precipitated, resuspended into 2× Laemmli loading buffer andanalyzed on SDS-PAGE.

The efficiency of intracellular EndoT processing was determined bycomparing the ratio between secreted glycosylated and non-glycosylated(or single GlcNAC-modified) hIFNβ observed for the transformants on theone hand and for the untransformed hIFNβ-producing strain on the otherhand (data not shown). Introduction of the fusion construct with the 240N-terminal Ste13p amino acids (fusion construct 2) did not result in achange in the ratio of glycosylated versus non-glycosylated hIFNβ.However, the expression of the fusion construct only containing thecytoplasmic and transmembrane domain of Ste13p (fusion construct 1), didresult in a change in the ratio: the amount of non-glycosylated (orsingle GlcNAc-modified) hIFNβ increased significantly when compared tothe untransformed hIFNβ production strain.

From the gel, it is also clear that there is some clonal variation (datanot shown): the least amount of glycosylated hIFNβ after 24 hours ofinduction was observed for clones 1, 4 and 7. Interestingly, at 48 hoursof induction, the gel pattern observed for these clones indicatesincreased cell lysis. Indeed, too high intracellular (Golgi) expressionof EndoT might result in serious cell stress due to severedeglycosylation of mannoproteins, thus weakening the cell wall. Growthof these clones in BMMY medium with 1 M of sorbitol as osmoticstabilizer, did not improve this result. These data indicate thatseveral clones can be checked in order to have a transformant with anice equilibrium between in vivo protein deglycosylation on the one handand resistance to lysis on the other hand.

Intracellular EndoT expression, however, also results in an extra bandon gel that is not present in the untransformed strain. This couldindicate that a fraction of the EndoT is released into the medium due toa proteolytic cleavage somewhere in the luminal domain, detaching itfrom the Ste13p localization signal. Activity of these proteolytic forms(both for fusion construct 1 and 2) was checked via a gel-shift analysisafter overnight incubation of some medium in NaOAc pH 5 withhyperglycosylated α-1,2-mannosidase as a test protein. The result ofthis analysis is shown in FIG. 2.

In lanes 1 and 4, no deglycosylation is observed on theα-1,2-mannosidase. The gel-shift analysis, however, indicates that bothproteolytic forms are active on α-1,2-mannosidase when incubated in theNaOAc buffer (whereas they only partially deglycosylate the co-expressedhIFNβ, still present in the medium that was used as EndoT enzymesource). So, although the proteolytic EndoT form derived from fusionconstruct 2 is active in the NaOAc buffer, no in vitro processing ofhIFNβ was observed in the medium itself (less efficient conditions).This, together with the tendency of the transformants of fusionconstruct 1 to undergo more cell lysis, indicates that the fusionprotein of EndoT with the first 140 amino acids of Ste13p is very likelyacting in vivo on the soluble hIFNβ and not in vitro in apost-secretorial way. Thus, it is possible to create viable cells thatproduce, deglycosylate and secrete proteins in vivo.

Moreover, although the in vitro activity of the proteolytic form is lowin the medium, this may primarily be due to two factors: the pH and thesubstrate. Indeed, the pH of the NaOAc buffer (5) is considerably lowerthan that of the medium (6.7). Furthermore, the assay also indicatesthat hIFNβ is a difficult substrate for EndoT, likely due to its compactfold and difficult accessibility of the glycans.

A subsequent test indeed indicated that EndoT is as efficient as EndoHin deglycosylating α-1,2-mannosidase in vitro. The deglycosylationcapacity of fusion construct 1 was also checked in a Pichia strainexpressing the soluble T. reesei (or H. jecorina) α-1,2-mannosidase. Theα-1,2-mannosidase is hyperglycosylated when produced by Pichia; hence,deglycosylation and the resulting higher mobility of the protein can beeasily evaluated via SDS-PAGE. Transformation of the expressionconstruct and selection and analysis of transformants was done asdescribed for the hIFNβ co-expression study (results not shown). Again,significant variation in between clones is observed when comparingdifferent EndoT transformants with the control sample (a Pichia strainsecreting the mannosidase without intracellular EndoT). After 24 hoursof induction, hyperglycosylation on the mannosidase is no longerpresent. Thus, if the conditions are suitable, glycoproteins may bedeglycosylated by EndoT, both in vivo and in vitro, in apost-secretorial way.

Conclusions:

Four forms of soluble EndoT were expressed in Pichia pastoris. Deletionof the 43 C-terminal amino acids results in a sharp decrease insecretion efficiency. Nevertheless, all four forms have the capacity todeglycosylate proteins after overnight incubation in NaOAc pH 5. EndoTis active on high-mannose but not on complex N-glycans and theefficiency of deglycosylation depends on the protein backbone to whichthe sugars are attached.

Expression of functional EndoT[FullSize] into the Pichia secretionpathway was successful when fusing the enzyme to the cytosolic andtransmembrane domain (first 140 N-terminal amino acids) of S. cerevisiaeSte13p, known to be localized in the yeast trans-Golgi network. Apartial in vivo deglycosylation of co-expressed hIFNβ (a difficultsubstrate for EndoT) and T. reesei, α-1,2-mannosidase was observed whilea fraction of the EndoT is also secreted into the medium as result ofintracellular proteolysis.

These results demonstrate that, e.g., Pichia pastoris can be used as aproduction platform for EndoT, which is an alternative for thecommercially available EndoH endoglucosaminidase. Moreover, EndoT can bea valuable tool for native deglycosylation of glycoproteins, e.g.,before crystallography or to remove undesired or immunogenicoligosaccharide chains, either via in vitro treatment with the enzyme orin vivo when co-expressed in a Pichia strain containing Golgi-localized(or possibly co-secreted) EndoT.

Since EndoT is able to trim high-mannose and hybrid N-glycans, itsexpression at the end of the Pichia secretion pathway should enable invivo clean-up of only partially humanized N-glycans (so not yet of thecomplex type) on recombinant proteins produced in a glyco-engineeredstrain. These non-complex glycans that are produced together withcomplex glycans in glyco-engineered strains are a known problem,especially because they are difficult to isolate from the glycoproteinswith complex glycosylation and may interfere with glycoprotein functionor immunogenicity. Since the amount of non-complex N-glycans is only afraction of the total modified N-glycan pool, moderate intracellularEndoT expression might already be sufficient to obtain a complete invivo clean-up of residual high-mannose and hybrid oligosaccharides.

Example 2 Production of Single GlcNAc-Modified Proteins in an EngineeredYeast Strain

Pichia pastoris strains are available, which have been extensivelyengineered to produce complex-type human bi- and multiantennaryN-glycans. These glycans can, moreover, be sialylated throughincorporation of a CMP-N-acetylneuraminic acid synthesis pathway in theyeast cell, together with a transporter for CMP-NANA from the cytoplasmto the Golgi lumen, and α-2,6-sialyltransferase.

As an example, we work with Pichia pastoris-expressing humaninterferon-beta as described in Example 1, in which the OCH1 gene hasbeen inactivated and in which Trichoderma reesei α-1,2-mannosidase,fused to a C-terminal HDEL-tag has been overexpressed, and in which alsohuman N-acetylglucosaminyltransferase I catalytic domain fused to theN-terminal region of S. cerevisiae Kre2p, Drosophila melanogasterMannosidase II catalytic domain fused to the N-terminal region of S.cerevisiae Mnn2p, human N-acetylglucosaminyltransferase II catalyticdomain fused to the N-terminal region of S. cerevisiae Mnn2p and afusion protein of S. cerevisiae Gal10p and the catalytic domain of humanbeta-1,4-galactosyltransferase, fused to the N-terminal region of S.cerevisiae Mnn2p, are all overexpressed. This strain producesbiantennary, bigalactosylated N-glycans, but also the intermediatesformed within this heterologously reconstructed pathway, i.e., somehigh-mannose glycans (including Man5GlcNAc2), and some hybrid glycans(including GalGlcNAcMan3-5) (PhD thesis Pieter Jacobs, Faculty ofSciences, Ghent University, 2008).

As described in Example 1, the 140 N-terminal amino acids of Ste13p,comprising the transmembrane region and the cytosolic domain known tocontain signals for Golgi-localization, were fused to the first aminoacid of Endo[FullSize] (=fusion construct 1), and this fusion constructis transformed to the glyco-engineered hIFNβ-producing strain describedin the previous paragraph. In this way, the large majority of EndoT isretained intracellularly in a late Golgi compartment and is active onthe N-glycans that pass this compartment. As the Kre2p and the Mnn2pproteins from which targeting signals used for localizing theglyco-engineering enzymes were derived, are known to localize to medialGolgi compartments in yeasts, these glyco-engineering enzymes havealready encountered the secreted glycoproteins before theseglycoproteins reach the endoT compartment and have thus converted theglycans on these secreted glycoproteins to complex-type biantennary,bigalactosylated structures, which are resistant to endoT hydrolysis.Nevertheless, the high-mannose and hybrid-type restproducts,intermediates of the built-in pathway, encounter endoT and arehydrolyzed, leaving only 1 GlcNAc residue on the protein perN-glycosylation site thus modified, and this happens before theglycoprotein is finally secreted from the cell.

Human IFNβ contains one N-glycosylation site and is produced by Pichiapastoris as a mixture of a glycosylated and a non-glycosylated form,which are easily distinguishable from one another on a 15%poly-acrylamide gel. For each construct, eight single clones are grownfor two days in 100 ml shake flasks containing 30 ml BMGY. Once highcell densities are reached, the expression of soluble hIFNβ andintracellular EndoT is transferred to BMMY medium. Proteins from 0.5 mlmedium, taken after 24 and 40 hours of induction, are DOC/TCAprecipitated, resuspended into 2× Laemmli loading buffer and analyzed onSDS-PAGE.

Comparing the ratio between secreted glycosylated and non-glycosylated(or single GlcNAc-modified) hIFNβ observed for the endoT transformantson the one hand and for the untransformed hIFNβ complex-typeglyco-engineered producing strain on the other hand, shows an increasein the non-glycosylated band when endoT was expressed, but this increaseis not as big as in Example 1, where high-mannose hIFNβ strains wereused, of which all N-glycans are sensitive to endoT.

The glycans remaining on secreted proteins, of which the major fractionis hIFNβ, are analyzed through deglycosylation of the proteins withpeptide-N-glycosidase F, labeling of the released glycans with APTS andprofiling of the glycans using capillary electrophoresis on aDNA-sequencer. As expected, the peaks corresponding to high-mannose andhybrid-type N-glycan structures add up to a significantly lowerpercentage of the total N-glycan mixture in the proteins secreted byendoT-engineered strain as compared to the non-endoT engineered strain,demonstrating that endoT engineering is efficient in removing thesehigh-mannose and hybrid-type glycans in vivo, thus improving thehomogeneity of glycosylation of therapeutic glycoproteins produced inthese glyco-engineered strains.

Example 3 Co-Secretion of Endoglucosaminidase and Glycoprotein byGlyco-Engineered Yeast

In this example, the setup of the experiment is entirely parallel to theone of Example 2, except that now we engineer the complex-typeglyco-engineered hIFNβ-producing strain with an expression construct forthe secretion of endoT protein, as detailed in Example 1 (where it wasdone in non-glyco-engineered yeast). In this fashion, endoT enzyme andhIFNβ are cosecreted in the culture medium of the yeast. As we bufferthe medium at pH=6.7 and as the pH optimum for endoT enzymatic activityis around 5.0, endoT is only very poorly active during the cultivationperiod, thus not affecting the physiology of the yeast. Upon completionof the hIFNβ production, the culture medium is harvested and the pH isshifted to 5.0 through double dialysis to NaAc pH=5.0 through a 3000 DaMWCO dialysis membrane. The preparation is subsequently incubated at 30°C. and samples are taken after 1 hour, 2 hours, 4 hours and 16 hours forprotein-linked N-glycan analysis through the method described above andfor SDS-PAGE analysis. The N-glycan analysis results demonstrate thathigh-mannose and hybrid-type N-glycans are progressively lost withincreasing incubation time, and the SDS-PAGE analysis concomittantlyshows an increase in the non-glycosylated hIFNbeta band, thusdemonstrating that cosecreted endoT can improve the homogeneity ofhIFNbeta toward complex-type N-glycans.

Example 4 Alternative Glycosylation Using Early Targeting ofEndoglucosaminidase

A Pichia pastoris strain expressing hIFNβ is used that is engineeredwith the medial-Golgi targeted fusion protein between the N-terminaltargeting signal of S. cerevisiae Mnn2p, the Gal10p catalytic domain andthe human beta-1,4-galactosyltransferase catalytic domain, as describedin Example 2.

EndoT is fused to the N-terminal targeting signal of Kre2p and the abovestrain is transformed with the expression construct for the Kre2p-EndoTfusion protein. In published studies, N-acetylglucosaminyltransferase Iwas targeted with this Kre2p targeting signal, and it was demonstratedthat this targeting puts the enzyme fused to it in a location so thatthe enzyme can convert its Man5GlcNAc2-glycoprotein substrate toGlcNAcMan5GlcNAc2-glycoprotein product before the glycoprotein reachesthe secretory system compartment where Mnn2p-fusion proteins arelocalized. Consequently, as hIFNβ leave the ER, it encounters theKre2p-EndoT fusion protein and its N-glycans are efficiently removed,resulting in hIFNβ being created that carries a single GlcNAc residueper N-glycosylation site. hIFNβ then moves on to theMnn2p-Gal10GalT-containing Golgi compartment, where the single GlcNAcresidues are recognized and modified with a β-1,4-Galactose residue,thus resulting in the formation of LacNAc structures, which are not asubstrate for any further endogenous yeast glycosyltransferases. Thus,hIFNβ modified with LacNAc N-glycans is secreted.

Using Western blotting of the secreted glycoproteins with the lectinRCA120, it is detected that the “differentially glycosylated” low-MWhIFNβ band is modified with terminal beta-galactose residues, whereasthis is not the case in the non-Kre2pEndoT-engineered control strain.This result is further confirmed to pre-treatment of the secretedproteins with β-1,4-galactosidase isolated from bovine kidney, resultingin a loss of the RCA120 lectin blotting signal.

As well-established in the art, LacNAc structures are excellentsubstrates for human alpha-2,6-sialyltransferase, and the sialylationpathway has been functionally incorporated in glyco-engineered Pichiastrains, which generated LacNAc structures using Mnn2p-targetedbeta-galactosyltransferase, as is also the case here. It is thus obviousto one skilled in the art that building in the published sialylationpathway in the LacNAc-N-glycan-producing Kre2pEndoT/Mnn2pGal10GalTstrain described above, will result in the secretion of glycoproteinsmodified with alpha2,6-sialylated LacNAc N-glycans. Therapeuticglycoproteins modified in this way are expected to be both veryhomogenous and have a long circulation time in vivo, as they would notbe recognized by hepatic and myeloid GlcNAc/Man or Gal/GalNAc receptors.

Alternatively, sialylation can be accomplished in vitro post-secretionthrough contacting of the LacNAc-N-glycan-modified glycoproteins withrecombinant alpha-2,6-sialyltransferase and CMP-NANA, using methods wellknown to those skilled in the art.

Example 5 Avoidance of Cell Lysis Upon Endoglucosaminidase Expressionand Glycan Profiles of Glyco-Engineered Yeast Strains

Pichia strains that overexpress both the test protein hIFNβ and EndoTcoupled to the cytoplasmic and transmembrane domain of Ste13p forintracellular expression were seen to give, after 48 hours of induction,a gel pattern, typical for cell lysis.

To avoid this cell lysis, probably due to weakening of the cell wall,resulting from too strong deglycosylation of its mannoproteins, analternative EndoT overexpressing strategy was used. Instead of thestrong AOX1 promoter, the AOX2 promoter was used to control theexpression of EndoT. AOX2 encodes a second AOX gene with 90% homology tothe AOX1 gene but is driven by a less active methanol-inducible AOX2promoter.

A new construct was made from pPICZSTE13CytoTMEndoT where the PAOX1 wasexchanged with the PAOX2 from pAOX2ZB from Invitrogen. GS115 strainsoverexpressing hIFNβ were transformed with the resulting plasmid.However, upon induction of these strains, no visible effect was seenfrom this EndoT expression, i.e., the ratio of glycosylated versusnon-glycosylated hIFNβ did not change (not shown; the efficiency ofintracellular EndoT processing was determined by comparing the ratiobetween secreted glycosylated and non-glycosylated hIFNβ observed forthe transformants on the one hand and for the untransformedhIFNβ-producing strain on the other hand).

Another approach is to overexpress EndoT extracellulary in the mediumtogether with the test protein. Therefore, another plasmid was madewhere the EndoT, fused to the pre-pro region of the S. cerevisiae alphamating factor for extracellular expression, was placed undertranscriptional control of the AOX2 promoter. Again, GS115 strainsoverexpressing hIFNβ were transformed with the resulting plasmid. Uponinduction of these strains, no visible effect was seen from this EndoTexpression, i.e., the ratio of glycosylated versus non-glycosylatedhIFNβ did not change (not shown).

This could be explained by the suboptimal pH for EndoT of the mediumbuffered to a pH 7. Therefore, the induced medium containing solubleEndoT and the hIFNβ test protein was incubated in a NaOAc pH 5 buffer at30° C. respectively for 1 hour, 3 hours and ON and compared to theuntreated medium of the eight different clones, as well as to the parentGS115 strain expressing hIFNβ, i.e., without EndoT (results not shown).

From these gels, we can conclude the soluble extracellular expressedEndoT indeed acts on the high mannose glycosylated hIFNβ, when the pH islowered to 5. Prolongation of the treatment results in a betterdeglycosylation. However, full deglycosylation appeared hard to achieve;this indicates again that hIFNβ is a difficult substrate for EndoT.

Glycan profiles were analyzed from clones 1, 4 and 6 and compared toGS115, with and without lowering of the pH to 5 in the induced medium.Extracellular soluble overexpression of EndoT seemed to already alterthe glycanprofile of the hIFNβ strains overexpressing EndoT compared toWT hIFNβ strains (FIG. 3). This might indicate cell stress.

The glycan patterns of the Pichia strains (the WT strain GS115 andMan5-glycoengineered strain) soluble overexpressing the different EndoTforms, N-terminal truncated, C-terminal truncated, C- and N-terminaltruncated or full size were also analyzed. The glycan profiles revealthat when overexpressing EndoT in the extracellular medium, the sugarcomposition of these strains is altered. Thus, the production of EndoTin the cells and its passage through the secretory pathway has aninfluence on the glycans of the strain (FIG. 4), so overexpressing EndoTin the yeast cells is not without consequences.

Next, the use of EndoT in the cleanup of glyco-engineered strains wastested. Since EndoT is able to trim high-mannose (and hybrid)N-glycans,its expression at the end of the Pichia secretion pathway should enablein vivo clean-up of only partially humanized N-glycans (so not yet ofthe complex type) on recombinant proteins produced in a glyco-engineeredstrain. Since the amount of non-complex N-glycans is only a fraction ofthe total modified N-glycan pool, moderate intracellular EndoTexpression might already be sufficient to obtain a complete in vivoclean-up of residual high-mannose and hybrid oligosaccharides.

Therefore, in vitro digests were performed on several differentglycoengineered strains producing GmCSF as a test protein. The strainsin order of engineering: GS115, Man5, GlcNAcMan5, GalGlcNAcMan5,GalGlcNAcMan3, Gal2GlcNAc2Man3 (named after glycosylation products).

These strains were methanol induced for 48 hours and sugars wereprepared and labeled. To remove the sugars from the glycoproteins, EndoTwas used in comparison with other glycosidases PNGaseF and EndoH. LikeEndoH, but unlike PNGase F, EndoT indeed seems unable to cut the complextype glycan Gal2Gn2Man3. In the case of GalGnMan3, the results are stillinconclusive and further experiments need to be done to check whetherEndoT could really help in cleaning up the heterogeneous glycosylationpattern. On the other hand, it is clear that EndoT acts on all thepresented sugar structures that contain a 6′ pentmannosyl group (FIG.5). In panel 5 of the GMCSF-GS 115 strain (FIG. 5F), a contaminatingpolymer is present, causing the aberrant glycan profile.

After EndoT/EndoH digest, another PNGaseF digest was performed on theimmobilized glycoproteins on the membrane, to check if there was stillsome glycoprotein left that could be deglycosylated with PNGaseF (panels5-6). Although some of these data are still inconclusive since productof an EndoT/H digest seems to still be present, it seems to confirm thatEndoT and EndoH, unlike PNGase F, are unable to cut the complex typeglycan Gal2Gn2Man3.

“Cleaning up” of the unwanted glycan structures thus evidently dependson the specificity of the endoglucosaminidase used. However, as EndoThydrolyzes high mannose glycans, a wild-type strain (i.e., that producesonly high mannose glycans) that overexpresses EndoT in large enoughamounts yields a strain that makes single GlcNAc-residues asN-glycosylation structures.

Example 6 In Vivo De-N-Glycosylation by Targeting of the Fungal endoTEnzyme to the Golgi-Apparatus of HEK293S GnTI^(−/−) Cells

To avoid in vitro deglycosylation, in vivo de-N-glycosylation in aHEK293S cell-line was implemented. Identification and cloning of afungal gene (Genbank Acc. No. CS423050) that encodes an endoH-typeendoglycosidase, denoted as endoT because it was cloned from thefilamentous fungi Trichoderma reesei (PhD thesis Ingeborg Stals, GhentUniversity, 2004), allows us to do so. The work is carried out in aglucosaminyltransferase I negative HEK cell-line (Reeves, Callewaert etal., PNAS (2002) 99:13419-13424). This cell-line almost exclusivelyproduces Man₅GlcNAc₂-N-glycans, which are hydrolyzed in the chitobiosebond by endoH-type endoglycosidases.

EndoT is secreted by T. reesei (now designated as Hypocrea jecorina),which is indicative for the fact that it is adapted to folding in theeukaryotic secretion pathway. In order not to interfere with thefunction of N-glycans in protein folding, endoT is targeted to thetrans-golgi/trans-golgi network.

Strategy

Targeting the endoT enzyme to the trans-golgi/TGN of the HEK293Scell-line is achieved by fusing the trans-golgi-targeting signal of agolgi-retained glycosyltransferase. Most golgi-residentglycosyltransferases are subject to proteolytic splicing in the stalkregion to a lesser or greater extent (Jaskiewicz, J. Biol. Chem.271(42):26395-26403 (1996)). The humanβ-galactoside-α-2,6-sialyltransferase (ST6GalI) or the humanganglioside-GM₂-synthase (GalNAcT)N-terminus is fused to the N-terminusof the full-length endoT enzyme. Theβ-galactoside-α-2,6-sialyltransferase (ST6GalI) has been characterizedbetter and its N-terminus is retained in the trans-golgi, but itcontains several cleavage sites and is probably subject to proteolyticprocessing (Kitazume-Kawaguchi et al., Glycobiology 9(12):1397-1406(1999)).

The GM2-synthase N-terminus is shorter: only the first 27 amino acidsseem to determine trans-golgi retention (Uliana et al., Traffic7:604-612 (2006)) and only contains one cathepsin-D splice site betweenamino acids 22 and 23 (GL-LYAST) (Jaskiewicz, J. Biol. Chem.271(42):26395-26403 (1996)). If too much cleaved, endoT fusion proteinis secreted; these sequences are mutated to a non-spliced sequence.

To evaluate proteolytic cleavage and targeting on the one hand and theefficiency of the in vivo de-N-glycosylation on the other, expressionconstructs for transient mammalian expression are made, using themammalian expression vector pCAGGS (Niwa et al., Gene 108:193-200(1991)). MYC-tagged constructs for the two fusion proteins allow forsubcellular localization experiments and to assess secretion.Subcellular localization experiments are carried out using an anti-MYCantibody immunofluoresence microscopy and a trans-golgi-targetingpHluorin construct (on the World Wide Web atbristol.ac.uk/synaptic/research/projects/mechanisms/phluorins.htm) as apositive control. Secretion of the MYC-tagged endoT protein is evaluatedby Western blot with an anti-MYC antibody and by using a MYC-taggedendoT without an N-terminal golgi-targeting sequence as a negativecontrol.

A soluble, secreted form of the glycoprotein hemagglutinin H3 is used tocotransfect to the HEK293S cell-line and allows evaluation of thede-N-glycosylating activity of the endoT fusion protein. Such ahemagglutinin coding sequence is also cloned into the pCAGGS vector. Ashemagglutinin is intracellularly deglycosylated by endoT, a shift inmolecular weight is observed on SDS-PAGE.

The best golgi-targeting signal is then used to make a final constructwith the chosen fusion protein. Constitutive, as well astetracycline-inducible, expression is envisaged.

For tetracycline-inducible expression, the pcDNA4/TO (Invitrogen) vectoris used. A stable cell-line is thus produced by selection with zeocin.The HEK293S GnTI−/− cell-line already contains a pcDNA6/TR construct,which encodes the Tet-repressor protein. This is constitutively andstably expressed and represses transcription from the pcDNA4/TO plasmid(Invitrogen) until tetracycline is added.

For constitutive expression, any mammalian expression vector containinga constitutive promoter and a selection marker (not blasticidin, alreadyin use for pcDNA6/TR) can be used.

Example 7 In Vivo De-N-Glycosylation of Glycoproteins by Targeting ofthe Fungal endoT Enzyme to the Secretory Pathway of Eukaryotic OrganismsStrains, Culture Conditions and Reagents.

Escherichia coli strains MC1061 were used for the amplification ofrecombinant plasmid DNA and grown in a thermal shaker at 37° C. inLuria-Broth (LB) medium supplemented with 100 μg/ml of carbenicillin or50 μg/ml of kanamycin, depending on the plasmids used.

Construction of pCAGGS-hST-endoT

The coding sequence for a fusion protein of which the N-terminal partconsists of the first 100 amino acids of the humanβ-galactoside-α-2,6-sialyltransferase (Genbank Acc. No. NM_(—)003032)and the C-terminal part consists of the full-size endoT, without signalsequence, was constructed as described (SEQ ID NO:14):

The endoT coding sequence with N- and C-terminus present, but withoutthe signal sequence, was amplified from pUC19endoT (full size) (see,above) by PCR with oligonucleotides “endoT.fusion.fw.251007” (TABLE 1)and “endoT.Bsu36I.rev.231007” (TABLE 1) and purified by agarose gelelectrophoresis.

The N-terminal part of the human β-galactoside-α-2,6-sialyltransferasewas amplified from a HepG2 library (Hepatoma cDNA library) by PCR witholigonucleotides “hSTGalI.XhoI.fw.231007” (TABLE 1) and“hSTGalI.fusion.rev.251007” (TABLE 1) and purified by agarose gelelectrophoresis.

The coding sequence for the fusion protein was amplified by fusion PCR,using these two PCR fragments as templates and with oligonucleotides“hSTGalI.XhoI.fw.231007” (TABLE 1) and “endoT.Bsu36I.rev.231007” (TABLE1). The resulting fragment was digested with Bsu36I and XhoI, andligated into a pCAGGS vector (Niwa et al., Gene 108:193-200 (1991)) thatwas also digested with Bsu36I and XhoI and treated with Calf IntestinePhophorylase (CIP). The insert in the resulting plasmid was sequencedusing oligonucleotides “pCAGGSF” and “pCAGGSRMARCO.”

Construction of pCAGGS-hST-endoT-myc

The coding sequence for a fusion protein of which the N-terminal partconsists of the first 100 amino acids of the humanβ-galactoside-α-2,6-sialyltransferase (Genbank Acc. No. NM_(—)003032)and the C-terminal part consists of the full-size endoT, without signalsequence and containing a C-terminal MYC-tag, was constructed asdescribed (SEQ ID NO:16):

The sequence encoding the fusion protein with a C-terminal MYC-tag wasamplified from pCAGGS-hST-endoT by PCR with oligonucleotides“hSTGalI.XhoI.fw.231007” (TABLE 1) and “endoT.Bsu36I.rev.231007” (TABLE1). The resulting fragment was purified by agarose gel electrophoresisand cloned into a pCR-bluntII-topo plasmid by topo-cloning, resulting inthe construct Topo-hST-endoT-myc. This construct was sequenced witholigonucleotides “SP6” (TABLE 1) and “T7” (TABLE 1) and the sequence ofthe fusion protein with C-terminal MYC-tag confirmed.

Topo-hST-endoT-MYC was digested with Bsu36I and XhoI, the fragmentcontaining the endoT construct was purified from the mix by agarose gelelectrophoresis and ligated into a pCAGGS vector (Niwa et al., Gene 108(1991), 193-200) that was also digested with Bsu36I and XhoI and treatedwith CIP.

Construction of pCAGGS-hGalNAcT-endoT

The coding sequence for a fusion protein of which the N-terminal partconsists of the first 27 amino acids of the humanUDP-GalNAc:lactosylceramide/GM3/GD3β-1,4-N-acetyl-galactosaminyltransferase (GalNAc-T or GA2/GM2/GD2synthase) (Genbank Acc. No. NM_(—)001478) and the C-terminal partconsists of the full size endoT, without signal sequence, wasconstructed as described (SEQ ID NO:18):

The endoT coding sequence with N- and C-terminus present but without thesignal sequence was amplified from pUC19endoT (full size) (see, above)by PCR with oligonucleotides “endoT.fushGalNacT.fw.231107” (TABLE 1) and“endoT.Bsu36I.rev.231007” (TABLE 1) and purified by agarose gelelectrophoresis.

The N-terminal part of the human GM2 synthase was amplified from a fetalbrain cDNA library (Dr. S. Ryckaert) by PCR with oligonucleotides“hGalNAcT.fw.XhoI.231107” (TABLE 1) and “hGalNacT.fus.rev.231107”(TABLE 1) and purified by agarose gel electrophoresis.

The coding sequence for the fusion protein was amplified by fusion PCR,using these two PCR fragments as templates and with oligonucleotides“hGalNAcT.fw.XhoI.231107” (TABLE 1) and “endoT.Bsu36I.rev.231007” (TABLE1). The resulting fragment was purified by agarose gel electrophoresisand cloned into a pCR-bluntII-topo plasmid by topo-cloning, resulting inthe construct Topo-GalNAcT-endoT. This construct was sequenced witholigonucleotides “SP6” (TABLE 1) and “T7” (TABLE 1) and the sequence ofthe fusion protein was confirmed.

Topo-hGalNAcT-endoT was digested with Bsu36I and XhoI, the fragmentcontaining the endoT construct was purified from the mix by agarose gelelectrophoresis and ligated into a pCAGGS vector (Niwa et al., Gene 108(1991), 193-200) that was also digested with Bsu36I and XhoI and treatedwith CIP.

Construction of pCAGGS-hGalNACT-endoT-myc

The coding sequence for a fusion protein of which the N-terminal partconsists of the first 27 amino acids of the humanUDP-GalNAc:lactosylceramide/GM3/GD3β-1,4-N-acetyl-galactosaminyltransferase (GalNAc-T or GA2/GM2/GD2synthase) (Genbank Acc. No. NM_(—)001478) and the C-terminal partconsists of the full-size endoT, without signal sequence and containinga C-terminal MYC-tag, was constructed as described (SEQ ID NO: 20):

The sequence encoding the fusion protein with a C-terminal MYC-tag wasamplified from Topo-hGalNAcT-endoT by PCR with oligonucleotides“hGalNAcT.fw.XhoI.231107” (TABLE 1) and “endoT.rev.myc.Bsu36I” (TABLE1). The resulting fragment was purified by agarose gel electrophoresisand digested with XhoI and Bsu36I and ligated into a pCAGGS vector (Niwaet al., Gene 108 (1991), 193-200) that was also digested with Bsu36I andXhoI and treated with CIP.

Cell Lines, Buffers and Antibodies

The Hek293S-Flt3 cell-line was obtained from Prof. S. Savvides(Department of Biochemistry and Microbiology, Faculty of Sciences,UGent). Cells were grown in DMEM/F12 medium (Gibco BRL, Invitrogen),supplemented with the following sterile supplements: 10% fetal calfserum, L-glutamin (0.3 g/L), penicillin G (100 u/mL), streptomycin (100μg/mL). Serum-free medium has the same formulation, with only the serumomitted. Lipofectamine 2000 was from Gibco BRL, Invitrogen. Tissueculture grade Tetracycline hydrochloride was from Sigma.

Phosphate buffered saline (PBS) is 137 mM NaCl, 2.7 mM KCl, 10 mMNa2HPO4.2H2O, 2 mM KH2PO4 and pH of 7.5. Chelating sepharose 6B beadswere from Pharmacia LKB.

The mouse monoclonal Penta-His IgG₁ antibody (BSA free) was from Qiagen;the mouse monoclonal anti-c-myc IgG₁ antibody was produced in-house; thesheep anti-mouse IgG₁ HRP-linked whole antibody was from Amersham, GEhealthcare.

Transient Transfection of endoT Constructs in Mammalian Cells

pCAGGS-hST-endoT, pCAGGS-hST-endoT-myc, pCAGGS-hGalNAcT-endoT andpCAGGS-hGalNAcT-endoT-myc were produced as described. These plasmids,and also the empty pCAGGS plasmid, were used to transiently transfectthe Hek293S-Flt3 cell-line. As a negative control, the cells were alsotransfected without DNA. Cells were seeded at 200,000 cells per well ina six-well plate two days prior to transfection so that they are atleast 85%-90% confluent at the day of transfection. Six hours prior totransfection, half of the medium was replaced by serum-free medium andthree hours prior to transfection, all medium (3 mL) was replaced by 2mL of serum-free medium. DNA lipoplexes were prepared by combining 4 μgof plasmid DNA with 10 μL of lipofectamine 2000 in 500 μL serum-freemedium and incubating for 20 minutes at room temperature. Afterincubation, the lipoplexes were added to the cells and incubatedovernight. The next morning, 1 mL of medium containing 30% serum wasadded to each well, to make a total serum concentration of 10%.

At the same time of transfection, 2 μg/mL Tetracycline Hydrochloride wasadded to each well to induce production of the Flt3 extracellular domain(secreted). 0.5 ml of the medium (without cells) was collected 48 and 72hours after transfection and stored at −20° C. for later analysis.

Sample Preparation of Medium Samples for Flt3 Detection

The medium samples containing BSA (from the fetal calf serum) werecleaned up using chelating sepharose 6B beads loaded with nickel ions.

Bead preparation: 500 μL beads were loaded with 1 mL of 100 mM nickelsulphate and incubated for 5 minutes at RT. They were spun down for 1minute at 500 g in a microcentrifuge and the supernatant was discarded.After this, they were washed with 1 mL of PBS, spun down for 1 minute at500 g and the supernatant was discarded. This wash step was repeatedfive times, and after the last wash, 500 μL of PBS was added.

Selective enrichment of his-tagged Flt3: to a sample of 250 μL, an equalamount of 2×PBS was added. 25 μL from the beads slurry (prepared asdescribed above) was added to this, and the mix was incubated on arotating platform for one hour.

After this, the beads were spun down for 1 minute at 500 g and thesupernatant was discarded. 0.5 mL of PBS was added to the beads, theywere spun down for 1 minute at 500 g and the supernatant was discarded.This wash step was done three times in total.

The beads were resuspended in 250 μL of PBS. Of the resulting samples,20 μL was taken, to which 10 μL of 3× Laemlli buffer with β-mercaptoethanol was added and the samples were cooked for 5 minutes.

Detection of Secreted Flt3 by Western Blot

After sample preparation, 30 μL of each sample was loaded onto a 10%SDS-PAGE gel and run. The gel was blotted semi-dry to a nitrocellulosemembrane and detection of the his-tagged Flt3 protein was performed witha primary penta-his antibody diluted 1/1000 and a secondary anti-mouseIgG1 diluted 1/5000.

Detection of Secreted endoT Constructs by Western Blot

The same medium samples were also used to assess secretion of(proteolytically cleaved) endoT fusion proteins. 10 μL of 3× Laemllibuffer with β-mercapto ethanol was added to 20 μL of the originalsamples, and these were run on a 10% SDS-PAGE gel. After blotting to anitrocellulose membrane, detection was performed using an anti-mycprimary antibody diluted 1/3000 and an anti-mouse secondary antibodydiluted 1/5000.

Results

The Hek293S-Flt3 was generated by the group of Prof. S. Savvides fromthe parental cell-line Hek293S-RicR, which produces almost exclusivelyMan5GlcNAc2 N-glycans. It is a stable transfectant line for thehis-tagged extracellular domain of the human Flt3 receptor; this proteingoes through the secretory pathway.

Transient Transfection of endoT Constructs into Mammalian Cells

The transfection protocol used allows us to transfect the cells with anefficiency of about 30%-40% (assessed by FACS, results not shown). Dailymicroscopic observation showed no significant cell death or a slowergrowth than the negative control well (transfection with no DNA) aftertransfecting any of the endoT fusion proteins or the empty pCAGGSplasmid.

Sample Preparation of Medium Samples for Flt3 Detection

Because of the presence of a high amount of bovine serum albumin (BSA)(runs at ˜66 kDa) in the samples, and the fact that the secreted,non-deglycosylated Flt3 receptor runs at about 70 kDa, immunodetectionof the Flt3 and especially detection of the deglycosylated forms of thisprotein, which run in the BSA area at a slightly lower molecular weightthan 70 kDa, is obscured by aspecific staining by the excess BSA andblocking of the actual Flt3 signal (see, FIG. 6). Therefore, it isconvenient to purify the Flt3 from the samples to a certain extent,using a cleanup step with nickel-loaded chelating sepharose beads. Thisstep selectively enriches the Flt3 molecules in the sample, since theyare his-tagged, and detection becomes possible.

Flt3 Western Blot: Processing by endoT

The secreted Flt3 extracellular domain contains nine putativeN-glycosylation sites (Rosnet et al., 1993). Up to this date, seven ofthese sites have been confirmed to be modified with N-glycans (personalcommunication, K. Verstraete). It is expected that removal of at leastsome of the glycans by the endoT fusion proteins will cause a band-shifton Western blot. FIG. 6 shows that this is indeed the case. Two dayspost-transfection and induction, some processing of the Flt3 produced bythe pCAGGS-hST-endoT and pCAGGS-hST-endoT-myc transfected cells can beobserved. After three days, no more fully glycosylated Flt3 can beobserved in any of the samples produced by endoT transfected cells (see,FIG. 6). The fact that the Flt3 bands originating from the cellstransfected with the myc-tagged endoT fusion proteins show the samebehavior as the ones from the non-myc-tagged endoT fusion proteintransfected cells, in both cases, is indicative for the fact that thec-myc tag does not seriously interfere with the function of the fusionproteins.

Detection of endoT Constructs by Western Blot

Both endoT fusion protein constructs were also tagged C-terminally witha c-myc tag. This allows for assessment of proteolytic processing andsubsequent secretion of the golgi-luminal domain of the endoT fusionproteins, which should then be detected in the supernatant by Westernblot. This is indeed the case for the endoT fused N-terminally to thetargeting domain of the human GM2-synthase (pCAGGS-hGalNAcT-endoT-myc)(not shown). Processing at a cathepsin D-like splice site (GL-LYAST)between amino acids 22 and 23 would give rise to a secreted fragment of˜39.1 kDa (non-glycosylated, myc-tagged form). The secreted fragment hasabout this size. The Coomassie stained SDS-PAGE gel shows small butclearly defined bands in the lanes loaded with supernatant samples frompCAGGS-hGalNAcT-endoT and pCAGGS-hGalNAcT-endoT-myc transfected cells,with a slight difference in MW, attributed to the presence or absence ofthe myc-tag (1.2 kDa) (not shown).

The endoT fused to the targeting domain of the humanβ-galactoside-α-2,6-sialyltransferase (hST) does not seem to be secretedin significant amounts, since no fragment can be detected on Westernblot three days after transfection with the pCAGGS-hST-endoT-mycplasmid. The first 27 amino acids of the fusion protein make up for thecytoplasmic and transmembrane domains. This means that, theoretically,anywhere between amino acid 27 and 100 (this is the portion of the hSTused), proteolytic splicing could occur and give rise to a fragment of38.6 kDa to 46.5 kDa. Even if N-glycans are present (four sites onendoT, no sites on hST targeting domain), taking into account thatN-glycans are of the Man5GlcNAc2 form, the protein would be outside ofthe BSA occluded area around 66 kDa (˜60-70 kDa) and thus would bedetected on Western blot. Also, the Coomassie stained SDS-PAGE gel showsno extra bands not present in the negative control lanes (transfectionwith empty pCAGGS) (not shown). All this indicates that the endoTprotein indeed remains inside the cell and thus is efficiently targeted.

TABLE 1 Primers: Name: Use: GCACTCGAGATGATTCACAC hSTGalI.XhoI.fw.231007Amplification hST6GalI N-terminal CAACCTGAAGAfragment, includes start codon and (SEQ ID NO: 22) XhoI siteTTAACGGGTACGTCCTTGTT hSTGalI.fusion.rev.251007 Amplification of hST6GalICCACACCTG N-terminal fragment, includes (SEQ ID NO: 23)sequence for fusion PCR to endoT fragment GCACTCGAGATGTGGCTGGGhGalNAcT.fw.XhoI.231107 Amplification of hGalNAcT CCGCCGGGN-terminal fragment, includes start (SEQ ID NO: 24) codon and XhoI siteTTAACGGGTACGGTGCTCGC hGalNacT.fus.rev.231107 Amplification of hGalNAcTGTACAGGAGCC N-terminal fragment, includes (SEQ ID NO: 25)sequence for fusion PCR to endoT fragment GAACAAGGACGTACCCGTTendoT.fusion.fw.251007 Amplification of endoT fragment, AAAGAACTGCAincludes sequence for fusion PCR to (SEQ ID NO: 26)hST6GalI N-terminal fragment CGCGAGCACCGTACCCGTTAendoT.fushGalNacT.fw.231107 Amplification of endoT fragment, AAGAACTGCAincludes sequence for fusion PCR to (SEQ ID NO: 27)hGalNAcT N-terminal fragment GCACCTGAGGTTAAGCGTTAendoT.Bsu36I.rev.231007 Amplification of endoT fragment, ACCATAGCGTAGincludes stop codon and Bsu36I site (SEQ ID NO: 28) GCACCTGAGGTTACAGATCTendoT.rev.myc.Bsu36I Amplification of endoT fragment,TCTTCAGAAATAAGCTTTTG includes sequence for MYC-tag, stopTTCAGCGTTAACCATAGCGT codon and Bsu36I site AGTAGTTGATGG (SEQ ID NO: 29)ACGTGCTGGTTATTGTGCTG pCAGGSF Forward sequencing primer for(SEQ ID NO: 30) endoT construct in pCAGGS vector GCCAGAAGTCAGATGCTCApCAGGSRMARCO Reverse sequencing primer for AGG (SEQ ID NO: 31)endoT construct in pCAGGS vector ATTTAGGTGACACTATAG SP6Forward sequencing primer for (SEQ ID NO: 32)inserts in the pCR-bluntII-topo plasmid AATACGACTCACTATAGGG T7Reverse sequencing primer for (SEQ ID NO: 33)inserts in the pCR-bluntII-topo plasmid

Sequence ID NO: 14XhoI site-Start codon-fusion protein-Stop codon-Bsu36I site ctcgagatgattcacaccaacctgaagaaaaagttcagctgctgcgtcctggtctttcttctgtttgcagtcatctgtgtgtggaaggaaaagaagaaagggagttactatgattcctttaaattgcaaaccaaggaattccaggtgttaaagagtctggggaaattggccatggggtctgattcccagtctgtatcctcaagcagcacccaggacccccacaggggccgccagaccctcggcagtctcagaggcctagccaaggccaaaccagaggcctccttccaggtgtggaacaaggacgtacccgttaaagaactgcagttgagagctgaaccaactgatttgcctaggcttatcgtttacttccagactactcacgactcttccaacagaccaatctccatgttgccattgatcactgagaagggtatcgctttgactcacttgatcgtttgttccttccacattaaccagggtggtgttgttcacttgaacgacttcccaccagatgatccacacttctacactttgtggaacgagactatcactatgaagcaggctggtgttaaggttatgggaatggttggtggtgctgctcctggttctttcaacactcagactttggactctccagactctgctactttcgagcactactacggtcaattgagagatgctatcgttaacttccagttggagggaatggatttggacgttgagcaaccaatgtcccaacaaggtatcgacagattgatcgctagattgagagctgatttcggtccagacttcttgattactttggctccagttgcttctgctttggaggactcctctaacttgtctggtttctcctacactgctttgcaacagactcagggtaacgacattgactggtacaacactcagttctactctggtttcggttctatggctgacacttccgactacgacagaatcgttgctaacggtttcgctccagctaaagttgttgctggtcagttgactactcctgaaggtgctggatggattccaacttcctccttgaacaacactatcgtttccttggtttccgagtacggtcaaatcggtggtgttatgggatgggagtacttcaattccttgccaggtggtactgctgaaccatgggagtgggctcaaatcgttactgagatcttgagaccaggattggttccagagctcaagattactgaggatgacgctgctagattgactggtgcttacgaagaatccgttaaggctgctgctgctgataacaagtccttcgttaagaggccttccatcaactactacgctatggttaacgcttaa cctcagg Sequence ID NO: 16XhoI site-Start codon-fusion protein-MYC tag-Stop codon-Bsu36I sitectcgagatgattcacaccaacctgaagaaaaagttcagctgctgcgtcctggtctttcttctgtttgcagtcatctgtgtgtggaaggaaaagaagaaagggagttactatgattcctttaaattgcaaaccaaggaattccaggtgttaaagagtctggggaaattggccatggggtctgattcccagtctgtatcctcaagcagcacccaggacccccacaggggccgccagaccctcggcagtctcagaggcctagccaaggccaaaccagaggcctccttccaggtgtggaacaaggacgtacccgttaaagaactgcagttgagagctgaaccaactgatttgcctaggcttatcgtttacttccagactactcacgactcttccaacagaccaatctccatgttgccattgatcactgagaagggtatcgctttgactcacttgatcgtttgttccttccacattaaccagggtggtgttgttcacttgaacgacttcccaccagatgatccacacttctacactttgtggaacgagactatcactatgaagcaggctggtgttaaggttatgggaatggttggtggtgctgctcctggttctttcaacactcagactttggactctccagactctgctactttcgagcactactacggtcaattgagagatgctatcgttaacttccagttggagggaatggatttggacgttgagcaaccaatgtcccaacaaggtatcgacagattgatcgctagattgagagctgatttcggtccagacttcttgattactttggctccagttgcttctgctttggaggactcctctaacttgtctggtttctcctacactgctttgcaacagactcagggtaacgacattgactggtacaacactcagttctactctggtttcggttctatggctgacacttccgactacgacagaatcgttgctaacggtttcgctccagctaaagttgttgctggtcagttgactactcctgaaggtgctggatggattccaacttcctccttgaacaacactatcgtttccttggtttccgagtacggtcaaatcggtggtgttatgggatgggagtacttcaattccttgccaggtggtactgctgaaccatgggagtgggctcaaatcgttactgagatcttgagaccaggattggttccagagctcaagattactgaggatgacgctgctagattgactggtgcttacgaagaatccgttaaggctgctgctgctgataacaagtccttcgttaagaggccttccatcaactactacgctatggttaacgctgaacaaaagcttatttctgaagaagatctg taa cctcaggSequence ID NO: 18XhoI site-Start codon-fusion protein-Stop codon-Bsu36I site Ctcgagatgtggctgggccgccgggccctgtgcgctctggtccttctgctcgcctgcgcctcgctggggctcctgtacgcgagcaccgtacccgttaaagaactgcagttgagagctgaaccaactgatttgcctaggatatcgtttacttccagactactcacgactcttccaacagaccaatctccatgttgccattgatcactgagaagggtatcgctttgactcacttgatcgtttgttccttccacattaaccagggtggtgttgttcacttgaacgacttcccaccagatgatccacacttctacactttgtggaacgagactatcactatgaagcaggctggtgttaaggttatgggaatggttggtggtgctgctcctggttctttcaacactcagactttggactctccagactctgctactttcgagcactactacggtcaattgagagatgctatcgttaacttccagttggagggaatggatttggacgttgagcaaccaatgtcccaacaaggtatcgacagattgatcgctagattgagagctgatttcggtccagacttcttgattactttggctccagttgcttctgattggaggactcctctaacttgtctggtttctcctacactgctttgcaacagactcagggtaacgacattgactggtacaacactcagttctactctggtttcggttctatggctgacacttccgactacgacagaatcgttgctaacggtttcgctccagctaaagttgttgctggtcagttgactactcctgaaggtgctggatggattccaacttcctccttgaacaacactatcgtttccttggtttccgagtacggtcaaatcggtggtgttatgggatgggagtacttcaattccttgccaggtggtactgctgaaccatgggagtgggctcaaatcgttactgagatcttgagaccaggattggttccagagctcaagattactgaggatgacgctgctagattgactggtgcttacgaagaatccgttaaggctgctgctgctgataacaagtccttcgttaagaggccttccatcaactactacgctatggttaacgcttaa cctcagg Sequence ID NO: 20XhoI site-Start codon-fusion protein-MYC tag-Stop codon-Bsu36I sitectcgagatgtggctgggccgccgggccctgtgcgctctggtccttctgctcgcctgcgcctcgctggggctcctgtacgcgagcaccgtacccgttaaagaactgcagttgagagctgaaccaactgatttgcctaggatatcgtttacttccagactactcacgactcttccaacagaccaatctccatgttgccattgatcactgagaagggtatcgctttgactcacttgatcgtttgttccttccacattaaccagggtggtgttgttcacttgaacgacttcccaccagatgatccacacttctacactttgtggaacgagactatcactatgaagcaggctggtgttaaggttatgggaatggttggtggtgctgctcctggttctttcaacactcagactttggactctccagactctgctactttcgagcactactacggtcaattgagagatgctatcgttaacttccagttggagggaatggatttggacgttgagcaaccaatgtcccaacaaggtatcgacagattgatcgctagattgagagctgatttcggtccagacttcttgattactttggctccagttgcttctgctttggaggactcctctaacttgtctggtttctcctacactgctttgcaacagactcagggtaacgacattgactggtacaacactcagttctactctggtttcggttctatggctgacacttccgactacgacagaatcgttgctaacggtttcgctccagctaaagttgttgctggtcagttgactactcctgaaggtgctggatggattccaacttcctccttgaacaacactatcgtttccttggtttccgagtacggtcaaatcggtggtgttatgggatgggagtacttcaattccttgccaggtggtactgctgaaccatgggagtgggctcaaatcgttactgagatcttgagaccaggattggttccagagctcaagattactgaggatgacgctgctagattgactggtgcttacgaagaatccgttaaggctgctgctgctgataacaagtccttcgttaagaggccttccatcaactactacgctatggttaacgctgaacaaaagcttatttctgaagaagatctg taa cctcagg

1. A eukaryotic cell comprising: a first exogenous polynucleotideencoding an endoglucosaminidase enzyme; and a second exogenouspolynucleotide encoding a glycoprotein.
 2. The eukaryotic cell of claim1, which does not express an endogenous endoglucosaminidase enzyme. 3.The eukaryotic cell of claim 1, which is selected from the groupconsisting of a yeast cell, a plant cell, a mammalian cell, an insectcell, an Hek293 cell, and a Pichia cell.
 4. The eukaryotic cell of claim1, which is a glyco engineered yeast cell and further comprises: atleast a third exogenous polynucleotide encoding at least one enzymeneeded for complex glycosylation, said enzyme selected from the groupconsisting of mannosidases and glycosyltransferases, other thanmannosyltransferases and phosphomannosyltransferases.
 5. The eukaryoticcell of claim 4, wherein the at least one enzyme needed for complexglycosylation is selected from the group consisting of Nacetylglucosaminyl transferase I, N acetylglucosaminyl transferase II,mannosidase II, galactosyltransferase, and sialyltransferase.
 6. Theeukaryotic cell of claim 5, which is a Pichia cell.
 7. The eukaryoticcell of claim 4, which is deficient in the functional expression of atleast one enzyme involved in the production of high mannose structures.8. The eukaryotic cell of claim 1, wherein the endoglucosaminidase is amannosyl glycoprotein endo beta N acetylglucosaminidase (E.C. 3.2.1.96).9. The eukaryotic cell of claim 1, wherein the glycoprotein is secretedby the cell.
 10. The eukaryotic cell of claim 9, wherein theendoglucosaminidase is also secreted by the cell.
 11. The eukaryoticcell of claim 1, wherein the endoglucosaminidase is operably linked toan endoplasmic reticulum or Golgi localization signal.
 12. Theeukaryotic cell of claim 11, wherein the endoplasmic reticulum or Golgilocalization signal is from a protein selected from the group consistingof Kre2p, Ste13p, GM2 synthase, α2,6, glycosyltransferase, and α2,6,sialyltransferase.
 13. The eukaryotic cell of claim 1, which isdeficient in an enzymatic activity needed for complex glycosylation,wherein the enzyme is selected from the group consisting of ERmannosidase I, glucosidase I, glucosidase II, N acetylglucosaminyltransferase I, N acetylglucosaminyl transferase II, mannosidase II, andwherein the cell is not capable of complex glycosylation ofglycoproteins.
 14. A plant comprising the eukaryotic cell of claim 1.15. A method for producing single GlcNAc modified proteins in aeukaryotic cell, the method comprising: providing an eukaryotic cellcomprising a first exogenous polynucleotide encoding anendoglucosaminidase enzyme and a second exogenous polynucleotideencoding a glycoprotein, in conditions suitable for expressing theendoglucosaminidase enzyme and the glycoprotein; and recovering theglycoprotein after it has been intracellularly or extracellularlycontacted with the endoglucosaminidase.
 16. The method according toclaim 15, wherein the intracellular contact with the endoglucosaminidaseoccurs in the Golgi or endoplasmic reticulum.
 17. The method accordingto claim 15, wherein the pH of the medium wherein the extracellularcontact takes place is adjusted for optimal enzymaticendoglucosaminidase activity.
 18. The method according to claim 15,wherein the eukaryotic cell is incapable of complex glycosylation ofglycoproteins.
 19. The method according to claim 15, further comprising:having the glycoprotein processed by a glycosyltransferase after theglycoprotein has been intracellularly or extracellularly processed withthe endoglucosaminidase.
 20. A method for producing proteins in a glycoengineered yeast cell and depleting proteins with high mannose typeglycosylation and/or hybrid type glycosylation, the method comprising:providing a glyco engineered yeast cell comprising a first exogenouspolynucleotide encoding an endoglucosaminidase enzyme, a secondexogenous polynucleotide encoding a glycoprotein, and a third exogenouspolynucleotide encoding at least one enzyme needed for complexglycosylation, the enzyme being selected from the group consisting ofmannosidases and glycosyltransferases other than mannosyltransferasesand phosphomannosyltransferases, in conditions suitable for expressingthe endoglucosaminidase enzyme, the glycoprotein and the enzyme neededfor complex glycosylation; and recovering the glycoprotein after it hasbeen intracellularly contacted with the enzyme needed for complexglycosylation and intracellularly or extracellularly contacted with theendoglucosaminidase.
 21. The method according to claim 20, wherein theintracellular contact with the endoglucosaminidase occurs in the Golgior endoplasmic reticulum, after contact with the at least one enzymeneeded for complex glycosylation.
 22. The method according to claim 20,wherein the intracellular contact with the endoglucosaminidase occurs inthe Golgi or endoplasmic reticulum, before contact with the at least oneenzyme needed for complex glycosylation.
 23. The method according toclaim 20, wherein the pH of the medium wherein the extracellular contacttakes place is adjusted for optimal enzymatic endoglucosaminidaseactivity.
 24. A eukaryotic cell comprising: a first exogenous nucleicacid molecule encoding an endoglucosaminidase enzyme; and a secondexogenous nucleic acid molecule encoding a glycoprotein, wherein theendoglucosaminidase enzyme deglycosylates the glycoprotein uponco-expression of the first and second exogenous nucleic acid molecule.25. A eukaryotic cell comprising: a first exogenous nucleic acidmolecule encoding Endo H; and a second exogenous nucleic acid moleculeencoding a glycoprotein.
 26. A eukaryotic cell comprising: a firstexogenous nucleic acid molecule encoding Endo M; and a second exogenousnucleic acid molecule encoding a glycoprotein.